JPWO2009044899A1 - Nucleic acids that control cell growth - Google Patents

Abstract

Cell growth inhibitors or growth promoters, diagnostic or therapeutic agents for diseases caused by cell growth abnormalities, apoptosis inducers, expression inhibitors or expression promoters for target genes of nucleic acids such as microRNA, cell growth inhibition The present invention relates to a method or a growth promotion method.

Description

  The present invention relates to a cell growth inhibitor or growth promoter using a nucleic acid, a diagnostic or therapeutic agent for a disease caused by abnormal cell growth, a cell growth suppression method or growth promotion method, and a nucleic acid target gene expression suppression. The present invention relates to a method or a method for promoting expression, a method for screening a cell growth inhibitor or a growth promoter.

  MicroRNA (miRNA), which is a kind of nucleic acid, is a small non-coding single-stranded RNA consisting of about 22 nucleotides that are not translated into protein, and is known to exist in many organisms including humans (Non-Patent Documents). 1, 2). MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. That is, a primary transcript, primary-microRNA (pri-miRNA), is first transcribed from a gene, and then in a stepwise process from pri-miRNA to mature microRNA, a precursor of about 70 bases with a characteristic hairpin structure -microRNA (pre-miRNA) is generated from pri-miRNA. Furthermore, mature microRNA is generated from pre-miRNA by Dicer-mediated processing (Non-patent Document 3).

Mature microRNAs are thought to be involved in post-transcriptional control of gene expression by binding complementarily to the target mRNA and suppressing translation of the mRNA, or by degrading the mRNA.
Although the mechanism by which microRNA suppresses the expression of target mRNA is not completely clarified, an outline has been elucidated by recent studies. MicroRNA binds to a partially complementary sequence in the 3'-untranslated region (3'-UTR) of the target mRNA, suppresses its translation, or suppresses expression by degrading the target mRNA To do. Although the above complementarity may not be perfect, it has been shown that complementarity of the 2nd to 8th bases from the 5′-terminal side of the microRNA is particularly important. Is sometimes referred to as a “seed sequence” (Non-patent Document 4). It has been shown that microRNAs with a common seed sequence suppress the expression of a common target mRNA even if other sequences are different. Therefore, RNA having microRNA-like activity can be designed by using a sequence complementary to the sequence present at the 3′-end of any mRNA as a seed sequence. Unlike siRNA, microRNA usually refers only to RNA that is “naturally present in cells”. Therefore, a microRNA-like sequence designed in this way may be particularly referred to as “artificial microRNA”.

  As of August 2007, the microRNA database miRBase (http://microrna.sanger.ac.uk/) contains 533 human and 5,071 microRNAs in all living species. Among the microRNAs expressed in mammals including humans, those known for their physiological functions include miR-181 (Non-patent Document 5) involved in blood cell differentiation and miR-375 (involved in insulin secretion). Non-patent document 6) is only a small part, and many of them have unclear physiological activity. However, studies using nematodes and Drosophila have revealed that microRNAs play various important roles in the development and differentiation of living organisms. There has been a report suggesting a relationship (Non-patent Document 7).

  Since microRNAs are involved in the control of the expression of various genes, abnormalities in microRNAs are expected to be involved in various human diseases. Research is progressing especially in cancer, and it has been reported that the expression of microRNA is different from that in normal tissues in many cancers, and that cancer can be classified by analyzing the expression profile of microRNA ( Non-patent document 8). It is also known that about half of the human microRNAs found so far are present at chromosomal abnormalities or fragile sites of chromosomes known in human cancer (Non-patent Document 9).

  Examples of cancer-microRNA relationships reported so far include the miR-15a / miR-16 cluster on chromosome 13q14, which is deleted in B-cell chronic lymphocytic leukemia (B-CLL), It is predicted that the deletion is one of the causes of B-CLL (Non-patent Document 10). In B-CLL, the expression of miR-29 and miR-181 is further reduced. One of the targets is known to be Tcl1 known as a proto-oncogene (Non-patent Document 11). In lung cancer, expression of Let-7, which is one of microRNAs, is decreased, and one of its targets is Ras known as a proto-oncogene (Non-patent Documents 12 and 13). Moreover, the expression of microRNAs such as miR-127 and miR-124a is reduced by hypermethylation modification of genes in cancer, and the target is Bcl6 or Cdk6 known as proto-oncogenes (Non-patent Document 14, 15). Many microRNAs have decreased expression in cancer cells, but there are also microRNAs in which gene amplification and overexpression are seen in cancer cells. For example, in a region where gene amplification is observed in malignant lymphoma, there are 6 types of microRNA clusters (miR-17-92). When this miRNA cluster gene is forcibly expressed in a model mouse of human B cell lymphoma, lymphoma It is known that the occurrence of the above is promoted (Non-patent Document 16). It has also been clarified that a gene called BIC, which has been regarded as a candidate oncogene that does not encode a protein overexpressed in Hodgkin lymphoma, encodes miR-155 (Non-patent Document 17).

As described above, many relations between cancer and microRNA have been reported in recent years, but many of them show abnormal expression in cancer cells, and reports showing the function of microRNA include Let-7 and miR-143. Report that the growth of cancer cell lines was inhibited by forced expression of miR-145 and cancer cells (Non-Patent Documents 18, 19, and 20), and cancer cells by forced expression of miR-16 and miR-34 There are few reports such as reports that the cell cycle of the strain was stopped (Non-Patent Documents 21 and 22), and reports that cell transformation was caused by forced expression of miR-372 and miR-373 (Non-Patent Document 23). . Cancer growth in an animal model by increasing microRNA expression by administering microRNA or its precursor from outside the body, or by reducing its expression by administering its antisense oligonucleotide Reported that tumor formation of the colon cancer cell line transplanted by miR-34a administration was suppressed (Non-patent Document 24), and tumor formation of the breast cancer cell line transplanted by miR-21 antisense administration. However, there are only a few reports (Non-patent Document 25).
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It is expected that new therapeutic agents and diagnostic agents will be developed by identifying microRNAs expressed in various human organs, analyzing their functions, and elucidating the relationship with diseases.
In particular, the discovery of microRNAs that cause cancer cell proliferation or suppression not only helps to understand the mechanisms of carcinogenesis, but also develops diagnostics and therapeutic agents for human cancer, as well as new diagnosis of cancer using them. It is expected to lead to methods and treatments. In addition to diseases other than cancer, cell proliferation abnormalities such as arteriosclerosis, rheumatoid arthritis, prostatic hypertrophy, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis, autoimmune diseases, etc. It is expected to contribute to the development of diagnostic and therapeutic agents for diseases caused by tissue hyperplasia and the like, and diagnostic and therapeutic methods using them.

  The object is to provide nucleic acids useful for the control of cell growth and methods for using them.

The present invention relates to the following (1) to (32).
(1) Cell growth inhibitor or growth promoter containing as an active ingredient any of the following nucleic acids (a) to (h):
(A) Nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321 (b) SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315 A nucleic acid consisting of a base sequence represented by any one of ˜2321, and a nucleic acid of 17 to 28 bases (c) represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261, and 2315-2321 (D) a nucleic acid comprising a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 939, 2057 to 2132, 2212 to 2261, and 2315 to 2321 Nucleic acids that hybridize with stringent conditions under complementary conditions (e) SEQ ID NOs: 1-939, 2057- 132, 2212 to 2261 and 2315 to 2321. Nucleic acids comprising the second to eighth base sequences of the base sequence represented by any of (2) SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 Nucleic acid consisting of a base sequence represented by any of the above (g) a base having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 Nucleic acid consisting of sequence (h) Nucleic acid that hybridizes under stringent conditions with a complementary strand of nucleic acid consisting of the base sequence represented by any one of SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314 and 2322 to 2328 ( 2) the nucleic acid is a microRNA or a microRNA precursor, (1) Cytostatic or growth promoting agent of cells described.
(3) A cell growth inhibitor or growth promoter comprising, as an active ingredient, a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid according to (1).
(4) A cell growth inhibitor or growth promoter comprising as an active ingredient the vector that expresses the nucleic acid according to (1) or (3).
(5) A cell growth inhibitor or growth promoter comprising as an active ingredient a substance that suppresses the expression of the gene having the target base sequence of the nucleic acid according to (1).
(6) A cell growth inhibitor or growth promoter comprising as an active ingredient a substance that promotes the expression of the gene having the target base sequence of the nucleic acid according to (1).
(7) The cell growth promoter or growth inhibitor according to (5) or (6), wherein the substance that suppresses or promotes expression is a nucleic acid.
(8) The cell growth promoter or growth inhibitor according to (7), wherein the nucleic acid is siRNA.
(9) A cell growth inhibitor or growth promoter containing the vector expressing the nucleic acid according to (7) as an active ingredient.
(10) A therapeutic agent for diseases caused by abnormal cell proliferation, comprising as an active ingredient the cell growth inhibitor or growth promoter according to any one of (1) to (9).
(11) A therapeutic agent for a disease caused by abnormal cell growth, comprising the nucleic acid according to any one of (1), (3) and (7) as an active ingredient.
(12) A diagnostic agent for a disease caused by abnormal cell proliferation, comprising as an active ingredient a reagent for detecting the expression level of the nucleic acid according to (1), a mutation of the nucleic acid, and a mutation of a genome encoding the nucleic acid.
(13) The therapeutic or diagnostic agent according to any one of (10) to (12), wherein the disease caused by abnormal cell proliferation is cancer.
(14) A method for treating a disease caused by abnormal cell proliferation, comprising administering an effective amount of the nucleic acid according to any one of (1), (3) and (7).
(15) A method for diagnosing a disease caused by abnormal cell proliferation, comprising detecting the expression level of the nucleic acid according to (1), a mutation of the nucleic acid, and a mutation of a genome encoding the nucleic acid.
(16) The treatment or diagnosis method according to (14) or (15), wherein the disease caused by abnormal cell proliferation is cancer.
(17) Use of the nucleic acid according to any one of (1), (3) and (7) for the manufacture of a therapeutic agent for a disease caused by abnormal cell proliferation.
(18) The use according to (17), wherein the disease caused by abnormal cell proliferation is cancer.
(19) An expression inhibitor for a target gene of the nucleic acid, comprising the nucleic acid according to (1) as an active ingredient.
(20) The nucleic acid target gene expression promoter according to (1), containing the nucleic acid according to (3) as an active ingredient.
(21) The nucleic acid target gene expression inhibitor or expression promoter according to (1), which contains the vector according to (4) as an active ingredient.
(22) A method for promoting cell growth or inhibiting cell growth, comprising using the nucleic acid according to (1) or (3).
(23) A method for promoting cell growth or inhibiting cell growth, comprising using the vector according to (4).
(24) A method for promoting cell growth or inhibiting cell growth, comprising using a substance that suppresses the expression of a target gene of the nucleic acid according to (1).
(25) A method for promoting cell growth or inhibiting cell growth, comprising using a substance that promotes the expression of a target gene of the nucleic acid according to (1).
(26) The method for promoting cell proliferation or inhibiting cell proliferation according to (24) or (25), wherein the substance that suppresses or promotes expression is a nucleic acid.
(27) The cell growth promotion method or growth suppression method according to (26), wherein the nucleic acid is siRNA.
(28) A method for promoting cell growth or a method for suppressing growth, which uses the vector expressing the nucleic acid according to (26).
(29) A method for suppressing the expression of a target gene of the nucleic acid, wherein the nucleic acid according to (1) is used.
(30) The method for promoting expression of a target gene of a nucleic acid according to (1), wherein the nucleic acid according to (3) is used.
(31) The method for suppressing expression or promoting expression of a target gene of nucleic acid according to (1), wherein the vector according to (4) is used.
(32) A method for screening a cell growth promoter or growth inhibitor, wherein the expression or function of the nucleic acid according to (1) is promoted or inhibited.

  According to the present invention, a cell growth inhibitor or growth promoter, a diagnostic or therapeutic agent for a disease caused by abnormal cell growth, an apoptosis inducer, an expression suppressor or expression promoter for a target gene of a nucleic acid such as microRNA, A method for inhibiting cell growth or a method for promoting growth can be provided.

FIG. 1 shows the results of Western blotting of Bcl2l1. The microRNA used in each lane is as follows. 1; miR-negacon # 2, 2; hsa-miR-337, 3; hsa-miR-491, 4; hsa-miR-342, 5; si-Bcl2l1. FIG. 2 shows the results of Western blotting of Birc5. The microRNA used in each lane is as follows. 1; miR-negacon # 2, 2; hsa-miR-337, 3; hsa-miR-491, 4; si-Birc5. FIG. 3 shows the results of Western blotting of Bcl9l. The microRNA used in each lane is as follows. 1; miR-negacon # 2, 2; hsa-miR-491, 3; hsa-miR-147, 4; hsa-miR-518b, 5; hsa-miR-432 *, 6; hsa-miR-16, 7 ; si-Bcl9l. FIG. 4 shows the results of a tumor formation experiment by transplantation of DLD-1 forcibly expressing hsa-miR-147,342. Each measurement point shows the average value and standard deviation of 12 transplant sites. FIG. 5 shows the results of a tumor formation experiment by transplantation of DLD-1 forcibly expressing hsa-miR-491. Each measurement point shows the average value and standard deviation of 12 transplant sites.

  The nucleic acid used in the present invention may be any molecule as long as nucleotides and molecules having functions equivalent to the nucleotides are polymerized, such as RNA that is a ribonucleotide polymer, DNA that is a deoxyribonucleotide polymer. , A polymer in which RNA and DNA are mixed, and a nucleotide polymer containing a nucleotide analog, and may be a nucleotide polymer containing a nucleic acid derivative. The nucleic acid in the present invention may be a single-stranded nucleic acid or a double-stranded nucleic acid. The double-stranded nucleic acid also includes a double-stranded nucleic acid in which one strand is hybridized under stringent conditions to the other strand.

  In the present invention, nucleotide analogues are used to improve or stabilize nuclease resistance, to increase affinity with complementary strand nucleic acids, to increase cell permeability, or to be visualized as compared to RNA or DNA. In addition, any molecule may be used as long as it is a molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA. Examples thereof include sugar moiety-modified nucleotide analogs and phosphodiester bond-modified nucleotide analogs.

The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to a part or all of the chemical structure of the nucleotide sugar. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged Nucleic Acid (BNA) having two circular structures, more specifically, 2′-position oxygen atom and 4′-position charcoal Locked artificial nucleic acid in which atoms are bridged via methylene (Locked Nucleic Acid) (LNA) , and ethylene bridged structure type artificial nucleic acid (Ethylene bridged nucleic acid) (ENA ) [Nucleic Acid Research, 32, e175 (2004)] is mentioned Furthermore, peptide nucleic acids (PNA) [Acc. Chem. Res., 32 , 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123 , 4653 (2001)], and peptides Ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122 , 6900 (2000)] and the like can be mentioned.

The phosphodiester bond-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical substance to a part or all of the chemical structure of a phosphodiester bond of a nucleotide. Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16 , 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].

  As a nucleic acid derivative, in order to improve nuclease resistance, to stabilize, to increase affinity with a complementary strand nucleic acid, to increase cell permeability or to be visualized as compared with a nucleic acid, a different chemical is used. Any molecule may be used as long as it is a molecule to which a substance is added, for example, 5′-polyamine addition derivative, cholesterol addition derivative, steroid addition derivative, bile acid addition derivative, vitamin addition derivative, Cy5 addition derivative, Cy3 addition derivative, 6-FAM Examples include addition derivatives, biotin addition derivatives, and the like.

Examples of the nucleic acid include nucleic acids represented by the following (a) to (k).
(A) Nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321 (b) SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315 A nucleic acid consisting of a base sequence represented by any one of ˜2321, and a nucleic acid of 17 to 28 bases (c) represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261, and 2315-2321 (D) a nucleic acid comprising a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 939, 2057 to 2132, 2212 to 2261, and 2315 to 2321 Nucleic acids that hybridize with stringent conditions under complementary conditions (e) SEQ ID NOs: 1-939, 2057- 132, 2212 to 2261 and 2315 to 2321, the nucleic acid (f) containing the second to eighth base sequences (f) (a) to (e), and the base sequence of the nucleic acid A nucleic acid comprising a nucleic acid containing a complementary base sequence, or a nucleic acid (g) (a) to (e) containing the double-stranded nucleic acid, and hybridizing with the nucleic acid under stringent conditions A double-stranded nucleic acid consisting of a nucleic acid to be soybean, or a double-stranded nucleic acid (h), (f) or (g) containing the double-stranded nucleic acid was linked with a spacer oligonucleotide consisting of 8 to 28 bases. A single-stranded nucleic acid having a hairpin structure, or a nucleic acid containing the single-stranded nucleic acid (i) consisting of a base sequence represented by any of SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 Nucleic acid j) Nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 940 to 2056, 2133-2211, 2262 to 2314 and 2322 to 2328 and having a nucleotide sequence of 90% or more (k) SEQ ID NOs: 940 to 2056 A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the base sequence represented by any of 2133-2211, 2262-2314, and 2322-2328.

  As the nucleic acid, microRNA is preferably used. Micro RNA refers to single-stranded RNA having a length of 17 to 28 bases. The surrounding genomic sequence containing the microRNA sequence has a sequence that can form a hairpin structure, and the microRNA can be excised from either strand of the hairpin. MicroRNAs complementarily bind to their target mRNA and suppress mRNA translation, or promote post-transcriptional control of gene expression by promoting mRNA degradation.

  Examples of the microRNA include human microRNA having a base sequence represented by any of SEQ ID NOs: 1 to 129, 803, 819, 916, 917, 925 and 2057 to 2114. Furthermore, as a microRNA having the same function as the human microRNA comprising the base sequence represented by any of SEQ ID NOs: 1 to 129, 803, 819, 916, 917, 925 and 2057 to 2114, an ortholog of the human microRNA It is represented by any one of SEQ ID NOs: 130 to 802, 822, 823, 826, 827, 829 to 840, 842, 843, 846 to 848, 850, 851, 2115 to 2122, 2212 to 2261, and 2315 to 2321. A nucleic acid having a base sequence can be mentioned. As a specific example, the ortholog of the human microRNA of SEQ ID NO: 1 is composed of the base sequence represented by SEQ ID NOs: 130 to 139 and 2212. Table 1 (Table 1-1 to Table 1-4) shows a correspondence table of microRNAs consisting of base sequences represented by SEQ ID NOs: 1 to 129, 803, 819, 916, 917, 925 and 2057 to 2114 and their orthologs. Show. The biological species shown at the top of Table 1 are as follows. has, Homo sapiens, human; mmu, Mus musculus, mouse; rno, Rattus norvegicus, rat; xla, Xenopus laevis, Xenopus laevis; xtr, Xenopus tropicalis; Mdo, Monodelphis domestica, gray porpoise, possum; age, Ateles geoffroyi, red spider monkey; lla, Lagothrix lagotricha, Humbold Woolley monkey; Gorilla gorilla, gorilla; ppa, Pan paniscus, bonobo; ptr, Pan troglodytes, chimpanzee; ppy, Pongo pygmaeus, orangutan; lca, Lemur catta, ring-tailed lemur; cgr, Cricetulus griseus, Chinese hamster; Ovis aries, sheep; ssc, Sus scrofa, pigs; dre, Danio rerio, zebrafish Fru, Fugu rubripes, tiger puffer; tni, Tetraodon nigroviridis, green puffer. Since human-derived microRNA and its ortholog have high sequence identity, they are considered to have similar functions. In addition, microRNAs that are subgrouped by the alphabet added to the end of the microRNA name also have similar functions because of their high sequence identity.

マイクロＲＮＡがその標的遺伝子のmRNAの翻訳を抑制するメカニズムとして、マイクロＲＮＡの5’末端側2〜8番目の塩基配列に対して相補的な塩基配列を有するmRNAは、マイクロＲＮＡ標的遺伝子として認識されることが知られている［Current Biology, 15, R458-R460 (2005)］。このメカニズムにより、該mRNAの発現がマイクロＲＮＡによって抑制される。従って、５’末端側2〜8番目が同じ塩基配列を有するマイクロＲＮＡは、同じmRNAの発現を抑制して同様の機能を有する。配列番号１〜１２９、８０３、８１９、９１６、９１７、９２５および２０５７〜２１１４のいずれかで表される塩基配列からなるマイクロＲＮＡと5’末端側2〜8番目が同じ塩基配列を有するマイクロＲＮＡとして、配列番号８０４〜８１８、８２０〜９１５、９１８〜９２４、９２６〜９３９および２１２３〜２１３２のいずれかで表される塩基配列からなる核酸をあげることができる。該マイクロＲＮＡの具体的な例としては、配列番号４で表される塩基配列からなるマイクロＲＮＡに対しては、配列番号８０４で表される塩基配列からなるマイクロＲＮＡをあげることができる。また、人工マイクロＲＮＡも、マイクロＲＮＡに含まれる。配列番号１〜１２９、８０３、８１９、９１６、９１７、９２５および２０５７〜２１１４で表される塩基配列からなるマイクロＲＮＡとその５’末端側2〜8番目が同じ塩基配列を有するマイクロＲＮＡの対応表を表２(表２−１から表２−４)に示す。共通のシード配列を有するマイクロＲＮＡは、その標的塩基配列が同一と考えられることから、同様の機能を有していると考えられる。

As a mechanism by which microRNA suppresses translation of mRNA of its target gene, mRNA having a base sequence complementary to the 2-8th base sequence on the 5 'end side of microRNA is recognized as a microRNA target gene. [Current Biology, 15 , R458-R460 (2005)]. By this mechanism, the expression of the mRNA is suppressed by the microRNA. Therefore, microRNAs having the same base sequence on the 2nd to 8th sides on the 5 ′ end side have the same function by suppressing the expression of the same mRNA. As a microRNA having a base sequence represented by any one of SEQ ID NOs: 1 to 129, 803, 819, 916, 917, 925, and 2057 to 2114, and a microRNA having the same base sequence on the second to eighth sides of the 5 ′ end And a nucleic acid having a base sequence represented by any one of SEQ ID NOs: 804 to 818, 820 to 915, 918 to 924, 926 to 939, and 2123 to 2132. As a specific example of the microRNA, for the microRNA consisting of the base sequence represented by SEQ ID NO: 4, microRNA consisting of the base sequence represented by SEQ ID NO: 804 can be exemplified. Artificial microRNA is also included in microRNA. Correspondence table between microRNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 1 to 129, 803, 819, 916, 917, 925 and 2057 to 2114 and microRNAs having the same nucleotide sequence on the second to eighth positions on the 5 ′ end Is shown in Table 2 (Tables 2-1 to 2-4). MicroRNAs having a common seed sequence are considered to have the same function because their target base sequences are considered identical.

上記の核酸としては、マイクロＲＮＡ前駆体もまた好ましく用いられる。マイクロＲＮＡ前駆体とは、上記核酸を含む約５０〜約２００塩基長、より好ましくは約７０〜約１００塩基長の核酸であり、かつ、ヘアピン構造を形成し得る核酸である。マイクロＲＮＡは、Dicerと呼ばれる蛋白質によるプロセシングを経て、マイクロＲＮＡ前駆体から生成される。

As the nucleic acid, a microRNA precursor is also preferably used. The microRNA precursor is a nucleic acid having a length of about 50 to about 200 bases, more preferably about 70 to about 100 bases including the nucleic acid, and can form a hairpin structure. MicroRNA is produced from a microRNA precursor through processing by a protein called Dicer.

  Examples of the microRNA precursor include a nucleic acid having the base sequence represented by SEQ ID NO: 940 or 941 for the human microRNA of SEQ ID NO: 1. For microRNAs consisting of the nucleotide sequences represented by any of SEQ ID NOs: 2-939, 2057-2132, 2212-2261 and 2315-2321, SEQ ID NOs: 942-2056, 2133-2211, 2262-2314 and A nucleic acid having a base sequence represented by any of 2322 to 2328 can be mentioned. Table 3 (Tables 3-1 to 3-17) shows a correspondence table between microRNAs and microRNA precursors in the present invention. In the present invention, the microRNA precursor includes an artificial microRNA precursor.

本発明において、配列番号１〜２３２８のいずれかで表される塩基配列と９０％以上の同一性を有する核酸とは、ＢＬＡＳＴ〔J. Mol. Biol., 215, 403 (1990)〕やＦＡＳＴＡ〔Methods in Enzymology, 183, 63 (1990)〕等の解析ソフトを用いて計算したときに、配列番号１〜２３２８のいずれかで表される塩基配列からなる核酸と少なくとも９０％以上、好ましくは９３％以上、より好ましくは９５％以上、さらに好ましくは９６％以上、特に好ましくは９７％以上、最も好ましくは９８％以上である核酸であることを意味する。

In the present invention, a nucleic acid having 90% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 1 to 2328 is BLAST [J. Mol. Biol., 215 , 403 (1990)] or FASTA [ Methods in Enzymology, 183 , 63 (1990)], etc., and at least 90% or more, preferably 93% of the nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 1 to 2328 More preferably, it means 95% or more, more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more.

The stringent conditions in the above are 7.5 mL, 1M Na ₂ HPO ₄ (pH 7.2) 0.6 mL, 10% SDS 21 mL, 50x Denhardt's solution 0.6 mL, 10 mg / Add the other strand labeled with ³² P-ATP to Hybridization buffer consisting of 0.3 mL of mL sonicated salmon sperm DNA, react at 50 ° C overnight, and then wash with 5xSSC / 5% SDS solution at 50 ° C for 10 minutes. Further, the condition is such that the signal can be detected by washing with 1 × SSC / 1% SDS solution at 50 ° C. for 10 minutes, and then removing the membrane and exposing it to an X-ray film.

  As a method for detecting the expression of a nucleic acid such as microRNA using a nucleic acid, any method can be used as long as it can detect the presence of a nucleic acid such as microRNA or a microRNA precursor in a sample. For example, (1 ) Northern hybridization, (2) dot blot hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay It is done.

  As a method for detecting a mutation in a nucleic acid such as microRNA using a nucleic acid, any method can be used as long as it can detect a mutation in the base sequence of a nucleic acid such as microRNA or a microRNA precursor in a sample. A method for detecting a heteroduplex formed by hybridization of a nucleic acid having a non-mutated base sequence and a nucleic acid having a mutant base sequence, or by directly sequencing a base sequence derived from a specimen, A method for detecting the presence or absence can be given.

  The vector for expressing nucleic acid may be any vector as long as it is designed to biosynthesize nucleic acid such as microRNA by being introduced into a cell and transcribed. Specific examples of vectors capable of expressing nucleic acids such as microRNA in cells include pCDNA6.2-GW / miR (Invitrogen), pSilencer4.1-CMV (Ambion), pSINsi-hH1 Examples thereof include DNA (manufactured by Takara Bio Inc.), pSINsi-hU6 DNA (manufactured by Takara Bio Inc.), pENTR / U6 (manufactured by Invitrogen), and the like.

As a method for suppressing the expression of a gene having a target base sequence of a nucleic acid such as microRNA (hereinafter referred to as a target gene), the activity of suppressing the expression of mRNA having a target base sequence of a nucleic acid such as microRNA is utilized. Any method may be used as long as it suppresses the expression of a gene having the target base sequence. In addition, suppression of expression here includes a case where the translation of mRNA is suppressed, and a case where the amount of protein translated from mRNA is reduced by cleaving or decomposing mRNA. Specific examples of substances that suppress the expression of mRNA having the target base sequence include nucleic acids such as siRNA and antisense oligonucleotides. The siRNA can be prepared based on the continuous sequence information of the mRNA [Genes Dev., 13 , 3191 (1999)]. The number of residues of the base constituting one strand of siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.

  The microRNA is a single-stranded RNA having a length of 17 to 28 bases comprising a sequence complementary to a continuous 7 base sequence present in the 3 ′ untranslated region of the mRNA of the target gene as the second to eighth base sequences. Some artificial microRNAs are also included. When the sequence including the sequence and extending it back and forth forms a hairpin structure, and the microRNA sequence is RNA that can be excised from any one strand of the hairpin structure in the cell by the microRNA biosynthetic pathway, The extended sequence is called an artificial microRNA precursor. Artificial microRNAs and artificial microRNA precursors can be designed using the method as described above using a gene whose expression is to be suppressed as a target gene.

The target base sequence of nucleic acid such as microRNA is a base sequence consisting of several bases recognized by nucleic acid such as microRNA in the present invention, and the expression of mRNA having the base sequence is such as microRNA in the present invention. A base sequence that is suppressed by a nucleic acid. The base sequence complementary to the second to eighth base sequences on the 5 ′ end side of the microRNA is that translation of mRNA having the base sequence is suppressed by the microRNA [Current Biology, 15 , R458-R460]. (2005)], a base sequence complementary to the 2-8th base sequence on the 5 ′ end side of a nucleic acid such as microRNA in the present invention can be mentioned as a target base sequence. For example, an mRNA containing a sequence that perfectly matches the 3 'UTR base sequence group of human mRNA by preparing a target sequence complementary to the 2-8th base sequence on the 5' end side of the microRNA Can be determined by selecting by a method such as a character string search. The 3'UTR base sequence group of human mRNA should be prepared using the genome sequence and gene position information that can be obtained from "UCSC Human Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway)". Can do. Specific examples of genes having a target base sequence of microRNA consisting of the base sequence represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321 include US National Biotechnology Table 4 with the names (Official Symbol and Gene ID) used in the EntreGene database (http://www.ncbi.nlm.nih.gov/Entrez/) of the National Center for Biotechnology Information (NCBI) The genes described in (Table 4-1 to Table 4-157) can be mentioned.

本発明においてマイクロＲＮＡなどの核酸を細胞で発現させる方法としては、細胞内に導入された際にマイクロＲＮＡなどをコードする遺伝子が発現する核酸を用いる方法があげられる。該核酸としてはＤＮＡやＲＮＡ、あるいはヌクレオチド類似体の他、これらのキメラ分子、あるいは該核酸の誘導体も用いることができる。具体的には、Pre-miR^TM miRNA Precursor Molecules（Ambion社製）やmiRIDIAN microRNA Mimics（GEヘルスケア社製）と同様に該核酸を設計し、該マイクロＲＮＡなどの核酸を細胞内で発現させることができる。マイクロＲＮＡを発現させる場合は、細胞内で最終的にマイクロＲＮＡができる状態になればいかなる方法でもよく、例えば、（１）マイクロＲＮＡ前駆体である一本鎖ＲＮＡを導入させる他、（２）マイクロＲＮＡそのもの、およびマイクロＲＮＡの相補鎖からなり、100%マッチの２本鎖からなるＲＮＡ、（３）マイクロＲＮＡがDicerに切断された後の状態を想定した２本鎖ＲＮＡを導入させる方法があげられる。こうした方法を使用した製品としては、それぞれ例えば、miCENTURY OX Precursor（B-Bridge社製）、miCENTURY OX siMature（B-Bridge社製）、miCENTURY OX miNatural（B-Bridge社製）をあげることができる。

In the present invention, a method for expressing a nucleic acid such as microRNA in a cell includes a method using a nucleic acid that expresses a gene encoding the microRNA when introduced into the cell. As the nucleic acid, in addition to DNA, RNA, or nucleotide analogues, these chimeric molecules or derivatives of the nucleic acids can also be used. Specifically, the nucleic acid is designed in the same manner as Pre-miR ^™ miRNA Precursor Molecules (Ambion) or miRIDIAN microRNA Mimics (GE Healthcare), and the nucleic acid such as the microRNA is expressed in the cell. Can do. When microRNA is expressed, any method may be used as long as microRNA can finally be produced in the cell. For example, (1) In addition to introducing single-stranded RNA as a microRNA precursor, (2) There is a method for introducing microRNA itself and RNA consisting of complementary strands of microRNA and 100% -matched double-stranded RNA, and (3) double-stranded RNA assuming the state after microRNA is cleaved into Dicer. can give. Examples of products using such a method include miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge).

  The method for synthesizing the nucleic acid used in the present invention is not particularly limited, and the nucleic acid can be produced by a known method using chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, etc. For example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. Examples of the enzymatic transcription method include transcription using a typical phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template.

Examples of a method for screening for a substance that promotes or suppresses the expression or function of the nucleic acid using the nucleic acid used in the present invention include, for example, introducing a vector that expresses the nucleic acid into a cell, and expressing an mRNA having the target base sequence. Alternatively, a method of screening for a substance that promotes or suppresses the function can be mentioned.
The medicament containing the nucleic acid used in the present invention as an active ingredient can be used for diagnosis or treatment of diseases caused by abnormalities such as cell proliferation or tissue hyperplasia. The nucleic acid can also be used as a cell growth inhibitor or growth promoter. Here, abnormal cell growth refers to a state in which cells are growing at a rate other than the normal growth rate in a living body.

Examples of diseases caused by abnormal cell proliferation or tissue hyperplasia include cancer, arteriosclerosis, rheumatoid arthritis, benign prostatic hyperplasia, and blood vessels after percutaneous transvascular coronary angioplasty. Restenosis, pulmonary fibrosis, glomerulonephritis, autoimmune disease and the like, preferably cancer can be mentioned.
Hereinafter, the present invention will be described in detail.
1. Method for detecting expression of nucleic acid expressed in cancer cell such as microRNA and microRNA precursor (1-1) Identification of microRNA Whether a small RNA sequence is a microRNA is determined by RNA (RNA), 9 , 277-279 (2003). For example, in the case of a low molecular weight RNA that has been newly acquired and whose base sequence has been determined, it can be performed as follows.

A peripheral genomic sequence obtained by extending the DNA sequence corresponding to the obtained low molecular RNA base sequence by about 50 nt to the 5 'end side and 3' end side, respectively, is obtained, and RNA predicted to be transcribed from the genomic sequence Predict secondary structure. As a result, when it has a hairpin structure and the base sequence of the small RNA is located in one strand of the hairpin, it can be determined that the small RNA is a microRNA. Genomic sequences are publicly available and are available, for example, from UCSC Genome Bioinformatics (http://genome.ucsc.edu/). In addition, various programs for secondary structure prediction have been published, such as RNAfold [Nucleic Acids Research, 31 , 3429-3431 (2003)], Mfold [Nucleic Acids Research, 31 , 3406-3415 (2003)], etc. Can be used. Further, existing microRNA sequences are registered in a database called miRBase at Sanger Center in the UK, and it can be determined whether or not they are identical to existing microRNAs based on whether or not they are identical to the sequences described here.
(1-2) Acquisition of sequence information of low molecular RNA Extracting total RNA from a human cancer cell line or a sample derived from a cancer patient, and using this RNA, low molecular RNA containing microRNA expressed in cancer cells or cancer tissues Can be obtained as follows.

As a method for obtaining low molecular weight RNA, specifically, low molecular weight RNA can be obtained by 15% polyacrylamide gel electrophoresis in accordance with the method described in Jeans & Development (Genes & Development), 15 , 188-200 (2000). The method of separating is mentioned. From this, 5'-terminal dephosphorylation, 3'-adapter ligation, phosphorylation, 5'-adapter ligation, reverse transcription, PCR amplification, concatamerization, and ligation to vector are sequentially cloned, and the clone is cloned. Can be determined. Alternatively, for example, according to the method described in Science, 294 , 858-862 (2001), 5′-adenylation, 3′-adapter ligation, 5′-adapter ligation, reverse transcription, PCR amplification, concatamerization In addition, after sequentially ligating to a vector, low molecular RNA can be cloned and the base sequence of the clone can be determined.

Further, according to the method described in Nucleic Acids Research, 34 , 1765-1771 (2006), 5′-end dephosphorylation, 3′-adapter ligation, phosphorylation, 5′-adapter ligation, reverse transcription, PCR amplification, microbeads By sequentially ligating to a vector, low molecular RNA is cloned, and the base sequence information of the low molecular RNA can be obtained by determining the base sequence by reading the base sequence of the microbead.

As another method for obtaining low molecular weight RNA, there is a method using a small RNA Cloning Kit (manufactured by Takara Bio Inc.).
(1-3) Method for detecting expression level of nucleic acid such as microRNA Examples of methods for detecting the expression level of nucleic acid such as microRNA and its precursor include (1) Northern hybridization and (2) dot blot high. Hybridization, (3) in situ hybridization, (4) quantitative PCR, (5) differential hybridization, (6) microarray, (7) ribonuclease protection assay and the like.

Northern blotting is a method in which sample-derived RNA is separated by gel electrophoresis, then transferred to a support such as a nylon filter, a probe labeled appropriately based on the base sequence of the nucleic acid is prepared, and hybridization and washing are performed. Thus, this is a method for detecting a band specifically bound to a nucleic acid. Specifically, for example, it can be performed according to the method described in Science, 294 , 853-858 (2001).

  The labeled probe is a base sequence of a nucleic acid used in the present invention, for example, a radioisotope, biotin, digoxigenin, a fluorescent group, a chemiluminescent group, etc. by a method such as nick translation, random priming or phosphorylation at the 5 ′ end. It can be prepared by incorporating it into DNA or RNA having a complementary sequence to LNA or LNA. Since the binding amount of the labeled probe reflects the expression level of the nucleic acid, the expression level of the nucleic acid can be quantified by quantifying the amount of bound labeled probe. Electrophoresis, membrane transfer, probe preparation, hybridization, and nucleic acid detection can be performed by the methods described in Molecular Cloning 3rd Edition [Cold Spring Harbor Press, (2001) Cold Spring Harbor, NY]. .

  In dot blot hybridization, RNA extracted from tissues and cells is spot-fixed on a membrane in a dotted manner, and then hybridized with a labeled polynucleotide that serves as a probe to detect RNA that specifically hybridizes with the probe. It is a method to do. As the probe, the same probe as in Northern hybridization can be used. Preparation of RNA, RNA spot, hybridization, and detection of RNA can be performed by the methods described in Molecular Cloning 3rd edition.

In situ hybridization uses paraffin or cryostat sections of tissues obtained from living organisms or immobilized cells as specimens, performs hybridization and washing steps with labeled probes, and observes the tissues and cells of nucleic acids by microscopic observation. This is a method for examining the distribution and localization in a cell [Methods in Enzymology, 254 , 419 (1995)]. As the probe, the same probe as in Northern hybridization can be used. Specifically, microRNA can be detected according to the method described in Nature Method, 3 , 27 (2006).

  In quantitative PCR, cDNA synthesized from a sample-derived RNA using a reverse transcription primer and a reverse transcriptase (hereinafter, the cDNA is referred to as a sample-derived cDNA) is used for measurement. As a reverse transcription primer used for cDNA synthesis, a random primer or a specific RT primer can be used. The specific RT primer refers to a primer having a sequence complementary to a nucleic acid and a base sequence corresponding to the surrounding genomic sequence.

For example, after synthesizing a specimen-derived cDNA, using this as a template, a template-specific design designed from a nucleotide sequence corresponding to a nucleic acid such as microRNA or a microRNA precursor and its surrounding genomic sequence, or a nucleotide sequence corresponding to a reverse transcription primer PCR is performed using a typical primer, a cDNA fragment containing the nucleic acid is amplified, and the amount of the nucleic acid contained in the sample-derived RNA is detected from the number of cycles until a certain amount is reached. As a template-specific primer, an appropriate region corresponding to the nucleic acid and the surrounding genomic sequence is selected, and consists of a sequence of 15 to 40 residues, preferably 20 to 30 residues at the 5 ′ end of the base sequence of the region. A set of DNA or LNA consisting of DNA or LNA and a sequence complementary to 15 to 40 residues, preferably 20 to 30 residues at the 3 ′ end can be used. Specifically, it can be performed according to the method described in Nucleic Acids Research, 32 , e43 (2004).

A specific RT primer having a stem-loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, the method described in Nucleic Acid Research, 33, e179 ( 2005) or can be performed using the TaqMan MicroRNA Assays (Applied Biosystems).
As yet another sample-derived cDNA synthesis method, a reverse transcription reaction can also be performed by adding a polyA sequence to a sample-derived RNA with polyA polymerase and using a base sequence containing an oligo dT sequence as a primer for reverse transcription. . Specifically, it can be performed using miScript System (Qiagen) or QuantiMir RT Kit (System Biosciences). Furthermore, by subjecting the sample-derived RNA or cDNA to hybridization to a filter or slide glass or silicon substrate or the like to which DNA or LNA corresponding to a base sequence containing at least one nucleic acid is immobilized, and washing. The variation in the amount of nucleic acid can be detected.

Examples of the method based on such hybridization include a method using differential hybridization [Trends Genet., 7 , 314 (1991)] and a microarray [Genome Res., 6 , 639 (1996)]. In either method, the difference in the amount of nucleic acid between the control sample and the target sample can be accurately detected by immobilizing an internal control such as a base sequence corresponding to U6 RNA on a filter or substrate. In addition, labeled cDNA synthesis using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) based on RNA derived from the control sample and the target sample, and two filters on one filter or one substrate. Accurate nucleic acid quantification can be performed by simultaneously hybridizing labeled cDNA. Furthermore, the nucleic acid can be quantified by directly labeling and hybridizing RNA derived from the control sample and / or the target sample. For example, using a microarray described in Proc. Natl. Acad. Sci. USA, 101 , 9740-9744 (2004), Nucleic Acid Research, 32 , e188 (2004), RNA, 13 , 151-159 (2007), etc. MicroRNA can be detected. Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion) and miRNA microarray kit (Agilent Technology).

In the ribonuclease protection assay, first, a promoter sequence such as T7 promoter and SP6 promoter is bound to the 3 ′ end of the nucleotide sequence corresponding to the nucleic acid or its surrounding genomic sequence, and labeled NTP (mixture of ATP, GTP, CTP, UTP) and Labeled antisense RNA is synthesized by an in vitro transcription system using RNA polymerase. The labeled antisense RNA is bound to the sample-derived RNA to form an RNA-RNA hybrid, and then digested with ribonuclease A that degrades only single-stranded RNA. The digest is subjected to gel electrophoresis, and RNA fragments protected from digestion by forming RNA-RNA hybrids are detected or quantified as nucleic acids. Specifically, it can be detected or quantified using mirVana miRNA Detection Kit (Ambion).
2. Synthesis of Nucleic Acids As described in 1 above, nucleic acids such as microRNAs and microRNA precursors that are expressed in cancer cells or cancer tissues, or whose expression is increased or decreased compared to normal tissues, were identified. Thereafter, not only RNA which is a polymer of ribonucleotides but also DNA which is a polymer of deoxyribonucleotides can be synthesized based on the base sequence. For example, the base sequence of DNA can be determined based on the base sequence of microRNA identified in 1 above. The base sequence of DNA corresponding to the base sequence of RNA can be uniquely determined by replacing U (uracil) contained in the RNA sequence with T (thymine). In addition, a polymer in which ribonucleotides and deoxyribonucleotides are mixed, a polymer containing nucleotide analogues, and a nucleic acid derivative can be synthesized in the same manner.

The method for synthesizing the nucleic acid is not particularly limited, and the nucleic acid can be produced by a known method using chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, etc. For example, synthesis with ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) Can do. Examples of the enzymatic transcription method include a transcription method using a typical phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template.
3. Method for detecting the function of a nucleic acid such as microRNA or microRNA precursor As a method for detecting the function of a nucleic acid such as microRNA, there is a method for detecting whether or not the translation of mRNA having a target base sequence is suppressed. be able to.

It is known that microRNA suppresses translation of mRNA containing the target base sequence in the 3 ′ terminal untranslated region (3′UTR) [Current Biology, 15 , R458-R460 (2005)]. Therefore, a DNA in which the target base sequence for the single-stranded RNA to be measured is inserted into the 3′UTR of an appropriate reporter gene expression vector is prepared, introduced into a host cell suitable for the expression vector, and single-stranded into the cell. By measuring the expression of a reporter gene when RNA is expressed, it can be detected whether or not it has a function of microRNA.

  The reporter gene expression vector may be any vector as long as it has a promoter upstream of the reporter gene and can express the reporter gene in the host cell. Any reporter gene can be used as the reporter gene.For example, firefly luciferase gene, Renilla luciferase gene, chloramphenicol acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, β -Lactamase gene, aequorin gene, green fluorescent protein gene and DsRed fluorescent gene can be used. Reporter gene expression vectors having such properties include, for example, psiCHECK-1 (Promega), psiCHECK-2 (Promega), pGL3-Control (Promega), pGL4 (Promega), pRNAi-GL ( Takara Bio), pCMV-DsRed-Express (CLONTECH) and the like. Single-stranded RNA can be expressed by the method described in 6 below.

Specifically, the function of single-stranded RNA as a microRNA can be detected as follows. First, host cells are cultured in a multi-well plate or the like to express a reporter gene expression vector having a target sequence and single-stranded RNA. Then, the reporter activity is measured, and the function of the single-stranded RNA as a microRNA is detected by measuring the reporter activity when the single-stranded RNA is expressed compared to the case where the single-stranded RNA is not expressed. can do.
4). Method for detecting mutations in nucleic acids such as microRNA and microRNA precursors As a method for detecting mutations in nucleic acids such as microRNAs and microRNA precursors, heterogeneous nucleic acids formed by hybridization of normal and mutant nucleic acids A method for detecting this strand can be used.

Methods for detecting heteroduplex include: (1) heteroduplex detection by polyacrylamide gel electrophoresis [Trends genet., 7 , 5 (1991)], (2) single strand conformation polymorphism analysis [Genomics, 16 , 325-332 (1993)], (3) Chemical cleavage of mismatches (CCM) [Human Genetics (1996), Tom Strachan and Andrew P. Read, BIOS Scientific Publishers Limited ], (4) Enzymatic cleavage method of mismatch [Nature Genetics, 9 , 103-104 (1996)], (5) Denaturing gel electrophoresis [Mutat. Res., 288 , 103-112 (1993)], etc. There are methods.

  The heteroduplex detection method by polyacrylamide gel electrophoresis is performed as follows, for example. First, using a specimen-derived DNA or a specimen-derived cDNA as a template, a primer smaller than 200 bp is amplified with a primer designed based on the genomic base sequence including the nucleic acid base sequence. When heteroduplexes are formed, the mobility is slower than homoduplexes without mutations, and they can be detected as extra bands. If the fragment is smaller than 200 bp, most insertions, deletions and substitutions of 1 base or more can be detected. Heteroduplex analysis is preferably performed on a single gel combined with single-strand conformation analysis described below.

  In single-strand conformation polymorphism analysis (SSCP analysis), using primers derived from the base sequence of the genome including the base sequence of the nucleic acid using the sample-derived DNA or the sample-derived cDNA as a template. The DNA amplified as a fragment smaller than 200 bp is denatured and electrophoresed in a native polyacrylamide gel. When DNA amplification is performed, the amplified DNA can be detected as a band by labeling the primer with an isotope or a fluorescent dye, or silver-staining the unlabeled amplification product. In order to clarify the difference from the wild type pattern, when a control sample is also run at the same time, a fragment having a mutation can be detected from the difference in mobility.

  In the mismatch chemical cleavage method (CCM method), DNA fragments amplified with primers designed based on the base sequence of the genome including the base sequence of the nucleic acid using the sample-derived DNA or the sample-derived cDNA as a template are used as isotopes or fluorescence in the nucleic acid. By hybridizing with a labeled nucleic acid incorporating a label and treating with osmium tetroxide, one strand of DNA at a position where a mismatch exists can be cleaved to detect a mutation. CCM is one of the most sensitive detection methods and can be applied to specimens of kilobase length.

Instead of the above osmium tetroxide, the mismatch can be cleaved enzymatically by combining RNase A with an enzyme involved in mismatch repair in cells such as T4 phage resol base and endonuclease VII.
In denaturing gradient gel electrophoresis (DGGE), DNA fragments amplified with primers designed based on the base sequence of the genome including the base sequence of the nucleic acid using the sample-derived DNA or the sample-derived cDNA as a template are chemically Electrophoresis is performed using a gel having a denaturant concentration gradient and a temperature gradient. The amplified DNA fragment moves in the gel to a position where it is denatured into a single strand and does not move after denaturation. Since there is a difference in the movement of the amplified DNA in the gel with and without the mutation, the presence of the mutation can be detected. In order to increase the detection sensitivity, a poly (G: C) terminal is preferably attached to each primer.

In addition, nucleic acid mutations can also be detected by directly determining and analyzing the base sequence of the sample-derived DNA or the sample-derived cDNA.
5. Methods for expressing nucleic acids such as microRNA and microRNA precursors Known microRNA sequences and their precursor sequences are registered in a database called miRBase at the Sanger Center in the UK. Nucleic acids such as RNA precursors can be made. It can also be prepared using the microRNA sequence obtained by the method described in 1.

  The nucleic acid can be expressed by using a vector that is biosynthesized by introduction into a cell and transcription. Specifically, based on the base sequence of the nucleic acid or the genomic base sequence containing the base sequence, a DNA fragment containing the hairpin portion is prepared and inserted downstream of the promoter of the expression vector to construct an expression plasmid. Next, the nucleic acid can be expressed by introducing the expression plasmid into a host cell suitable for the expression vector.

  As the expression vector, a vector that can replicate autonomously in a host cell or can be integrated into a chromosome and contains a promoter at a position where a gene containing a nucleic acid base sequence can be transcribed is used. Any promoter can be used as long as it can be expressed in the host cell. For example, the RNA polymerase II (pol II) promoter and the RNA polymerase III (pol III) system which is a transcription system of U6 RNA and H1 RNA. A promoter etc. can be mentioned. Examples of the pol II promoter include the IE (immediate early) gene promoter of cytomegalovirus (human CMV), the early promoter of SV40, and the like. Examples of expression vectors using them include pCDNA6.2-GW / miR (Invitrogen), pSilencer 4.1-CMV (Ambion) and the like. Examples of pol III promoters include U6 RNA, H1 RNA, and tRNA gene promoters. Examples of expression vectors using them include pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), and pENTR / U6 (Invitrogen).

Alternatively, a gene containing a nucleic acid base sequence is inserted downstream of a promoter in a viral vector to construct a recombinant viral vector, the vector is introduced into a packaging cell to produce a recombinant virus, and the nucleic acid base A gene containing the sequence can also be expressed.
The packaging cell may be any cell as long as it can replenish the deficient protein of the recombinant viral vector deficient in any of the genes encoding the proteins required for viral packaging, such as human kidney. Derived HEK293 cells, cells derived from mouse fibroblasts NIH3T3, and the like can be used. Proteins supplied by packaging cells include mouse retrovirus-derived gag, pol, env, etc. for retrovirus vectors, and HIV virus-derived gag, pol, env, vpr, vpu for lentiviral vectors. , vif, tat, rev, nef, etc., adenovirus vectors such as E1A and E1B derived from adenovirus, and adeno-associated virus vectors such as Rep (p5, p19, p40), Vp (Cap), etc. Can be used.

In addition to using an expression vector, the nucleic acid used in the present invention can be directly introduced into a cell without using a vector. As a nucleic acid used in this method, in addition to DNA, RNA, or nucleotide analogues, these chimeric molecules or derivatives of the nucleic acids can be used. Specifically, nucleic acids such as microRNA and microRNA precursors can be expressed in the same manner as Pre-miR ^™ miRNA Precursor Molecules (Ambion) and miRIDIAN microRNA Mimics (GE Healthcare). When microRNA is expressed, any method can be used as long as microRNA can finally be produced in the cell. For example, (1) In addition to introducing single-stranded RNA as a microRNA precursor, (2) There is a method of introducing microRNA itself and RNA consisting of a complementary strand of microRNA and 100% -matched double-stranded RNA, and (3) double-stranded RNA assuming a state after microRNA is cleaved into Dicer. can give. Products using these methods include miCENTURY OX Precursor (B-Bridge), miCENTURY OX siMature (B-Bridge), and miCENTURY OX miNatural (B-Bridge).
6). Methods for suppressing the activity of nucleic acids such as microRNAs and microRNA precursors Nucleic acids such as microRNAs and microRNA precursors are antisense technologies [Bioscience and Industry, 50 , 322 (1992), Chemistry, 46, 681 ( 1991), Biotechnology, 9 , 358 (1992), Trends in Biotechnology, 10 , 87 (1992), Trends in Biotechnology, 10 , 152 (1992), Cell Engineering, 16 , 1463 (1997)], Triple Helix Technology [ Trends in Biotechnology, 10 , 132 (1992)], ribozyme technology [Current Opinion in Chemical Biology, 3 , 274 (1999), FEMS Microbiology Reviews, 23 , 257 (1999), Frontiers in Bioscience, 4 , D497 (1999), Chemistry & Biology, 6 , R33 (1999), Nucleic Acids Research, 26 , 5237 (1998), Trends In Biotechnology, 16 , 438 (1998)], Decoy DNA method [Nippon Rinsho-Japanese Journal of Clinical Medicine, 56 , 563 (1998), Circulation Research, 82 , 1023 (1998), Experimental Nephrology, 5 , 429 (1997), Nippon Rinsho-Japanese Journal of Clinical Medicine, 54 , 2583 (1996)], or siRNA (short interfering RNA) can be used to suppress the activity.

Antisense refers to a nucleic acid that can specifically hybridize a nucleic acid having a base sequence complementary to the base sequence of a certain target nucleic acid to suppress the expression of the target nucleic acid. As the nucleic acid used for antisense, in addition to DNA, RNA or nucleotide analogs, these chimeric molecules or derivatives of the nucleic acids can also be used. Specifically, antisense can be produced and expression can be suppressed by following the method described in Nature, 432 , 226 (2004) and the like. Moreover, expression of nucleic acids such as microRNA and microRNA precursors can be suppressed in the same manner as Anti-miR ^™ miRNA Inhibitors (Ambion) and miRIDIAN microRNA Inhibitors (GE Healthcare).

The siRNA is a short double-stranded RNA containing a base sequence of a certain target nucleic acid and can suppress the expression of the target nucleic acid by RNA interference (RNAi). sequence of the siRNA, the literature of the nucleotide sequence [Genes Dev., 13, 3191 (1999)] targeting can be appropriately designed based on the conditions. For example, siRNA can be prepared by synthesizing and annealing two RNAs having a sequence of 19 bases selected and a sequence obtained by adding TT to the 3 ′ end of each complementary sequence and annealing. Further, by inserting a DNA corresponding to the selected 19-base sequence into a siRNA expression vector such as pSilencer 1.0-U6 (Ambion) or pSUPER (OligoEngine), the expression of the gene can be suppressed. A vector expressing siRNA can be prepared. The siRNA that suppresses nucleic acid such as microRNA used in the present invention may be any siRNA that can suppress the activity of the nucleic acid, but SEQ ID NOs: 1-939, 2057-2132, 2212-2261, and 2315-2321. SiRNA designed from the continuous sequence information after the 9th base in each of the base sequences represented by any of the above is preferred. The number of residues of the base constituting one strand of siRNA is preferably 17 to 30 residues, more preferably 18 to 25 residues, still more preferably 19 to 23 residues.

  Expression of microRNAs or microRNA precursors expressed in cancer cells can be suppressed using antisense or siRNA specific to nucleic acids such as microRNAs or microRNA precursors expressed in cancer cells. For example, by administering antisense DNA or siRNA specific to the microRNA or the microRNA precursor, the activity of the microRNA is suppressed and the action of the microRNA or microRNA precursor in cancer cells is controlled. be able to.

  In addition, in the case of patients with abnormal expression of microRNAs or precursors thereof expressed in cancer cells, the function of cancer cells can be achieved by administering to the patient an antisense oligonucleotide or siRNA specific for the microRNAs or precursors thereof. Can be controlled to treat diseases that develop due to the above abnormal expression. That is, the antisense oligonucleotide or siRNA specific to the microRNA or a precursor thereof is useful as a therapeutic agent for diseases caused by abnormal growth of cancer cells.

When an antisense oligonucleotide or siRNA specific to a nucleic acid such as microRNA or a precursor thereof is used as the above therapeutic agent, the antisense oligonucleotide or siRNA alone or the nucleic acid encoding them is a retroviral vector, After being inserted into an appropriate vector such as an adenovirus vector or an adeno-associated virus vector, it can be administered as a pharmaceutical preparation according to the conventional method described in 9 below.
7). Method of suppressing gene function using nucleic acid such as microRNA or microRNA precursor As a method of suppressing function or expression of target gene of nucleic acid, nucleic acid such as microRNA is used for expression of mRNA having target base sequence. Any method may be used as long as it uses the inhibitory activity. For example, by expressing a nucleic acid such as microRNA and increasing the amount of nucleic acid such as microRNA in the cell, it is possible to suppress the translation of mRNA having the target sequence and to suppress the expression of the gene. . The nucleic acid can be expressed by the method described in 5 above. Examples of mRNA having a target base sequence of a nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 939, 2057 to 2132, 2212 to 2261, and 2315 to 2321 include, for example, the genes shown in Table 4 above, respectively. Groups can be exemplified.

Moreover, the function of this target gene can be suppressed using siRNA with respect to the target gene shown in Table 4.
8). Method for screening a substance that promotes or suppresses expression or function of nucleic acid using nucleic acid such as microRNA or microRNA precursor Using nucleic acid to promote expression or function of nucleic acid such as microRNA or its precursor Substances to be suppressed can be screened. For example, by selecting a base sequence to be screened from the base sequence of a nucleic acid and utilizing a cell that expresses a nucleic acid having the base sequence, the expression or function of the selected microRNA or its precursor is promoted or Substances to be suppressed can be screened.

As a cell expressing microRNA and a microRNA precursor used for screening, in addition to cancer cells, a vector expressing a nucleic acid having the base sequence is used as a host such as an animal cell or yeast as described in 5 above. A transformed cell obtained by introduction into a cell, a cell in which a nucleic acid having the base sequence is directly introduced without using a vector, and the like can also be used.
Specific screening methods include methods that use changes in the expression level of nucleic acids such as microRNAs or their precursors to be screened as indicators, as well as mRNAs that have nucleic acid target sequences such as microRNAs, and encoded by them. A method using the change in the expression level of the gene product as an index can be mentioned.
(A) Screening method using as an index a change in the expression level of a nucleic acid such as a microRNA or a precursor thereof as a target for screening The test substance is contacted with a cell expressing the nucleic acid, and the expression level of the selected nucleic acid is determined. Using the change as an indicator, a substance that promotes or suppresses nucleic acids such as expression of microRNA and its precursor is obtained. The expression level of the nucleic acid can be detected by the method described in 3 above.
(B) Screening method using as an index the change in the expression level of mRNA having a nucleic acid target sequence such as microRNA to be screened and the gene product encoded by it Contact test cells with cells expressing the mRNA Thus, a substance that promotes or suppresses the expression or function of nucleic acids such as microRNA and its precursor is obtained using changes in the expression level of mRNA having the target sequence of the selected nucleic acid and the gene product encoded thereby as an index. Alternatively, a DNA in which the target sequence of a nucleic acid such as microRNA is inserted into the 3′UTR of an appropriate reporter gene expression vector is prepared, introduced into a host cell suitable for the expression vector, and a test substance is contacted with the cell, Using a change in the expression level of the reporter gene as an indicator, a substance that promotes or suppresses the expression or function of nucleic acids such as microRNA and its precursor is obtained.

Selection of the target sequence can be performed by the method described in 7 above, such as a microRNA comprising a base sequence represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321 Examples of mRNA having a nucleic acid target sequence include the gene groups shown in Table 4 above.
9. Cell growth inhibitors or growth promoters using nucleic acids such as microRNAs and microRNA precursors Nucleic acids such as microRNAs and microRNA precursors, and nucleic acids having a complementary base sequence to the target sequence By controlling the expression of a gene having, it can be used as a cell growth inhibitor or growth promoter.

Examples of the cell growth inhibitor include the following nucleic acids (a) to (h).
(A) SEQ ID NOs: 1-3, 5-7, 11, 14, 15, 17, 24, 25, 29-33, 35, 37, 39-54, 56-58, 61-63, 66-68, 70 72-82, 84, 85, 87-112, 114-121, 125, 128-167, 193-219, 263-278, 306-324, 357-368, 476-492, 522-557, 561-564 572-576, 578-683, 693-714, 735-743, 746-751, 754-762, 765-803, 853-855, 888-891, 899-912, 914-926, 930-939, 2058 2060, 2061, 2063-2065, 2067-2071, 2073-2078, 2080, 2082, 2084-2092, 2096-2 108, 2110, 2111, 2113, 2117, 2120-2122, 2125-2128, 2132, 2212, 2213, 2216, 2219, 2220, 2227-2229, 2231-2237, 2240-2243, 2248-2253, 2255-2261, Nucleic acid consisting of the base sequence represented by any of 2317 and 2318 (b) SEQ ID NOs: 1-3, 5-7, 11, 14, 15, 17, 24, 25, 29-33, 35, 37, 39- 54, 56-58, 61-63, 66-68, 70, 72-82, 84, 85, 87-112, 114-121, 125, 128-167, 193-219, 263-278, 306-324, 357-368, 476-492, 522-557, 561-564, 572-576, 578- 683, 693-714, 735-743, 746-751, 754-762, 765-803, 853-855, 888-891, 899-912, 914-926, 930-939, 2058, 2060, 2061, 2063 2065, 2067-2071, 2073-2078, 2080, 2082, 2084-2092, 2096-2108, 2110, 2111, 2113, 2117, 2120-2122, 2125-2128, 2132, 2212, 2213, 2216, 2219, 2220, Nucleic acid of 17 to 28 bases containing a nucleic acid consisting of the base sequence represented by any of 2227 to 2229, 2231 to 2237, 2240 to 2243, 2248 to 2253, 2255 to 2261, 2317 and 2318 (c) SEQ ID NO: 1 -3, 5-7, 11, 14, 15, 17, 24, 25, 29-33, 35, 37, 39-54, 56-58, 61-63, 66-68, 70, 72-82, 84 85, 87-112, 114-121, 125, 128-167, 193-219, 263-278, 306-324, 357-368, 476-492, 522-557, 561-564, 572-576, 578 ~ 683, 693-714, 735-743, 746-751, 754-762, 765-803, 853-855, 888-891, 899-912, 914-926, 930-939, 2058, 2060, 2061, 2063 -2065, 2067-2071, 2073-2078, 2080, 2082, 2084-2092, 2096-2108, 211 2111, 2113, 2117, 2120-2122, 2125-2128, 2132, 2212, 2213, 2216, 2219, 2220, 2227-2229, 2231-2237, 2240-2243, 2248-2253, 2255-2261, 2317 and 2318 (D) SEQ ID NOs: 1-3, 5-7, 11, 14, 15, 17, 24, 25, 29- 33, 35, 37, 39-54, 56-58, 61-63, 66-68, 70, 72-82, 84, 85, 87-112, 114-121, 125, 128-167, 193-219, 263-278, 306-324, 357-368, 476-492, 522-557, 561-564, 5 2 to 576, 578 to 683, 693 to 714, 735 to 743, 746 to 751, 754 to 762, 765 to 803, 853 to 855, 888 to 891, 899 to 912, 914 to 926, 930 to 939, 2058, 2060, 2061, 2063-2065, 2067-2071, 2073-2078, 2080, 2082, 2084-2092, 2096-2108, 2110, 2111, 2113, 2117, 2120-2122, 2125-2128, 2132, 2212, 2213, 2216, 2219, 2220, 2227 to 2229, 2231 to 2237, 2240 to 2243, 2248 to 2253, 2255 to 2261, 2317, and 2318 Nucleic acids that hybridize under various conditions (e) SEQ ID NOs: 1-3, 5-7, 11, 14, 15, 17, 24, 25, 29-33, 35, 37, 39-54, 56-58, 61- 63, 66-68, 70, 72-82, 84, 85, 87-112, 114-121, 125, 128-167, 193-219, 263-278, 306-324, 357-368, 476-492, 522-557, 561-564, 572-576, 578-683, 693-714, 735-743, 746-751, 754-762, 765-803, 853-855, 888-891, 899-912, 914- 926, 930-939, 2058, 2060, 2061, 2063-2065, 2067-2071, 2073-2078, 2080, 2082, 084 to 2092, 2096 to 2108, 2110, 2111, 2113, 2117, 2120 to 2122, 2125 to 2128, 2132, 2212, 2213, 2216, 2219, 2220, 2227 to 2229, 2231 to 2237, 2240 to 2243, 2248 Nucleic acids comprising the 2nd to 8th base sequences of the base sequences represented by any of 2253, 2255 to 2261, 2317 and 2318 (f) SEQ ID NOs: 940 to 945, 947, 948, 952, 953, 956 to 958, 960, 968, 969, 973-978, 980, 982, 984-1001, 1003-1005, 1008-1009, 1012-1014, 1016, 1018-1029, 1031, 1033-1059, 1064-1069, 1073, 10 6-1133, 1163 to 1188, 1232 to 1259, 1288 to 1309, 1343 to 1354, 1479 to 1495, 1529 to 1566, 1572 to 1576, 1585 to 1589, 1591 to 1709, 1719 to 1744, 1765 to 1777, 1780 to 1788, 1791-1829, 1898-1900, 1998-2001, 2009-2050, 2052-2056, 2134, 2136, 2137, 2139-2143, 2145-2151, 2153-2158, 2160, 2162, 2164-2172, 2176- 2188, 2190, 2191, 2193, 2197, 2200 to 2202, 2205 to 2207, 2221, 2262 to 2265, 2268, 2271 to 2273, 2281 to 2283, 2 285 to 2292, 2295 to 2297, 2302 to 2306, 2308 to 2314, 2324 and 2325. Nucleic acid (g) SEQ ID NOs: 940 to 945, 947, 948, 952, 953, 956 to 958, 960, 968, 969, 973-978, 980, 982, 984-1001, 1003-1005, 1008-1009, 1012-1014, 1016, 1018-1029, 1031, 1033-1059, 1064-1069, 1073, 1076 to 1133, 1163 to 1188, 1232 to 1259, 1288 to 1309, 1343 to 1354, 1479 to 1495, 1529 to 1566, 1572 to 1576, 1585 to 1589, 1591 to 1709, 1719 to 1744, 176 5-1777, 1780-1788, 1791-1829, 1898-1900, 1998-2001, 2009-2050, 2052-2056, 2134, 2136, 2137, 2139-2143, 2145-2151, 2153-2158, 2160, 2162, 2164 to 2172, 2176 to 2188, 2190, 2191, 2193, 2197, 2200 to 2202, 2205 to 2207, 2221, 2262 to 2265, 2268, 2271 to 2273, 2281 to 2283, 2285 to 2292, 2295 to 2297, 2302 2306, nucleic acid consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by any one of 2306, 2308 to 2314, 2324 and 2325 (h) SEQ ID NOs: 940 to 945, 947, 948, 952, 953, 956-958, 960, 968, 969, 973-978, 980, 982, 984-1001, 1003-1005, 1008-1009, 1012-1014, 1016, 1018-1029, 1031, 1033-1059, 1064 to 1069, 1073, 1076 to 1133, 1163 to 1188, 1232 to 1259, 1288 to 1309, 1343 to 1354, 1479 to 1495, 1529 to 1566, 1572 to 1576, 1585 to 1589, 1591 to 1709, 1719 to 1744, 1765 to 1777, 1780 to 1788, 1791 to 1829, 1898 to 1900, 1998 to 2001, 2009 to 2050, 2052 to 2056, 2134, 2136, 2137, 2139 to 2143, 145 to 2151, 2153 to 2158, 2160, 2162, 2164 to 2172, 2176 to 2188, 2190, 2191, 2193, 2197, 2200 to 2202, 2205 to 2207, 2221, 2262 to 2265, 2268, 2271 to 2273, 2281 A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid consisting of the nucleotide sequence represented by any one of 2283, 2285-2292, 2295-2297, 2302-2306, 2308-2314, 2324 and 2325.

Examples of the cell growth promoter include the nucleic acids listed in (i) to (p) below.
(I) SEQ ID NOs: 4, 8 to 10, 12, 13, 16, 18 to 23, 26 to 28, 34, 36, 38, 55, 59, 60, 64, 65, 69, 71, 83, 86, 113 122-124, 126, 127, 168-192, 220-262, 279-305, 325-356, 369-475, 493-521, 558-560, 565-571, 577, 684-692, 715-734 , 744, 745, 752, 753, 763, 764, 804-852, 856-887, 892-898, 913, 927-929, 2057, 2059, 2062, 2066, 2072, 2079, 2081, 2083, 2093-2095 2109, 2112, 2114 to 2116, 2118, 2119, 2123, 2124, 2129 to 2131 , 2214, 2215, 2218, 2221 to 2226, 2230, 2238, 2239, 2244 to 2247, 2254, 2315, 2316 and 2319 to 2321 nucleic acid (j) SEQ ID NOs: 4 and 8 -10, 12, 13, 16, 18-23, 26-28, 34, 36, 38, 55, 59, 60, 64, 65, 69, 71, 83, 86, 113, 122-124, 126, 127 168-192, 220-262, 279-305, 325-356, 369-475, 493-521, 558-560, 565-571, 577, 684-692, 715-734, 744, 745, 752, 753 763, 764, 804-852, 856-887, 892-898, 913, 927-929, 20 57, 2059, 2062, 2066, 2072, 2079, 2081, 2083, 2093-2095, 2109, 2112, 2114-2116, 2118, 2119, 2123, 2124, 2129-2131, 2214, 2215, 2218, 2221-2226, Nucleic acid having 17 to 28 bases (k) SEQ ID NOs: 4 and 8 to 8 containing a nucleic acid consisting of the base sequence represented by any of 2230, 2238, 2239, 2244 to 2247, 2254, 2315, 2316 and 2319 to 2321 10, 12, 13, 16, 18-23, 26-28, 34, 36, 38, 55, 59, 60, 64, 65, 69, 71, 83, 86, 113, 122-124, 126, 127, 168-192, 220-262, 279-305, 325-356, 3 69-475, 493-521, 558-560, 565-571, 777, 684-692, 715-734, 744, 745, 752, 753, 763, 764, 804-852, 856-887, 892-898, 913, 927-929, 2057, 2059, 2062, 2066, 2072, 2079, 2081, 2083, 2093-2095, 2109, 2112, 2114-2116, 2118, 2119, 2123, 2124, 2129-2131, 2214, 2215, 2218, 2221 to 2226, 2230, 2238, 2239, 2244 to 2247, 2254, 2315, 2316 and a nucleic acid comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence (1 ) SEQ ID NO: 4, 8-10, 12, 13, 16, 18-23, 26-28, 34, 36, 38, 55, 59, 60, 64, 65, 69, 71, 83, 86, 113, 122-124, 126, 127, 168-192, 220-262, 279-305, 325-356, 369-475, 493-521, 558-560, 565-571, 577, 684-692, 715-734, 744, 745, 752, 753, 763, 764, 804-852, 856-887, 892-898, 913, 927-929, 2057, 2059, 2062, 2066, 2072, 2079, 2081, 2083, 2093-2095, 2109, 2112, 2114- 2116, 2118, 2119, 2123, 2124, 2129-2131, 2214, 22 5, 2218, 2221 to 2226, 2230, 2238, 2239, 2244 to 2247, 2254, 2315, 2316 and 2319 to 2321 and hybridize under stringent conditions with the complementary strand of the nucleic acid consisting of the nucleotide sequence Nucleic acid (m) SEQ ID NOs: 4, 8 to 10, 12, 13, 16, 18 to 23, 26 to 28, 34, 36, 38, 55, 59, 60, 64, 65, 69, 71, 83, 86 113, 122-124, 126, 127, 168-192, 220-262, 279-305, 325-356, 369-475, 493-521, 558-560, 565-571, 577, 684-692, 715 -734, 744, 745, 752, 753, 763, 764, 804-852, 856-887, 89 -898, 913, 927-929, 2057, 2059, 2062, 2066, 2072, 2079, 2081, 2083, 2093-2095, 2109, 2112, 2114-2116, 2118, 2119, 2123, 2124, 2129-2131, 2214 , 2215, 2218, 2221 to 2226, 2230, 2238, 2239, 2244 to 2247, 2254, 2315, 2316 and 2319 to 2321, a nucleic acid comprising the second to eighth base sequences (n ) SEQ ID NOs: 946, 949-951, 954, 955, 959, 961-967, 970-972, 979, 981, 983, 1002, 1006, 1007, 1010, 1011, 1015, 1017, 1030, 1032, 1060-1 63, 1070-1072, 1074, 1075, 1134-1162, 1189-1231, 1260-1287, 1310-1342, 1355-1478, 1496-1528, 1567-1571, 1577-1584, 1590, 1710-1718, 1745 1764, 1778, 1779, 1789, 1790, 1830-1897, 1901-1997, 2002-2008, 2051, 2133, 2135, 2138, 2144, 2152, 2159, 2161, 2163, 2173-2175, 2189, 2192, 2194- 2196, 2198, 2199, 2203, 2204, 2208 to 2210, 2266, 2267, 2270, 2274 to 2280, 2284, 2293, 2294, 2298 to 2301 2307, 2322, 2323 and nucleic acid consisting of the base sequence represented by any of 2326 to 2328 (o) SEQ ID NOs: 946, 949 to 951, 954, 955, 959, 961 to 967, 970 to 972, 979, 981, 983, 1002, 1006, 1007, 1010, 1011, 1015, 1017, 1030, 1032, 1060-1063, 1070-1072, 1074, 1075, 1134-1162, 1189-1231, 1260-1287, 1310-1342, 1355 1478, 1496-1528, 1567-1571, 1577-1584, 1590, 1710-1718, 1745-1764, 1778, 1779, 1789, 1790, 1830-1897, 1901-1997, 2002-200 2051, 2133, 2135, 2138, 2144, 2152, 2159, 2161, 2163, 2173-2175, 2189, 2192, 2194-2196, 2198, 2199, 2203, 2204, 2208-2210, 2266, 2267, 2270, 2274 Nucleic acid (p) SEQ ID NO: consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any of ˜2280, 2284, 2293, 2294, 2298-2301, 2307, 2322, 2323 and 2326-2328 946, 949-951, 954, 955, 959, 961-967, 970-972, 979, 981, 983, 1002, 1006, 1007, 1010, 1011, 1015, 1017, 1030, 1032, 1060-1063 1070 to 1072, 1074, 1075, 1134 to 1162, 1189 to 1231, 1260 to 1287, 1310 to 1342, 1355 to 1478, 1496 to 1528, 1567 to 1571, 1577 to 1584, 1590, 1710 to 1718, 1745 to 1764, 1778, 1779, 1789, 1790, 1830-1897, 1901-1997, 2002-2008, 2051, 2133, 2135, 2138, 2144, 2152, 2159, 2161, 2163, 2173-2175, 2189, 2192, 2194-2196, 2198, 2199, 2203, 2204, 2208 to 2210, 2266, 2267, 2270, 2274 to 2280, 2284, 2293, 2294, 2298 to 2301, 230 A nucleic acid that hybridizes under stringent conditions with a complementary strand of a nucleic acid comprising the nucleotide sequence represented by any of 7, 2322, 2323, and 2326 to 2328.

As the nucleic acid, a microRNA or a microRNA precursor is preferably used.
Examples of cell growth promoters include nucleic acids having a base sequence complementary to the base sequences of the nucleic acids described in (a) to (h) above, and examples of cell growth inhibitors include the above (i) to ( and a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid described in p). In addition, the above-mentioned nucleic acids and vectors expressing nucleic acids complementary thereto can also be used as cell growth inhibitors or growth promoters.

  On the other hand, a substance that suppresses the expression of a target gene of a nucleic acid such as the above microRNA, or a substance that promotes the expression of the target gene can also be used as a cell growth inhibitor or a growth promoter. As this substance, a nucleic acid or a vector expressing it can also be used. Examples of substances that suppress the expression of the target gene include siRNA against the mRNA of the target gene and antisense to the target gene. Substances that promote the expression of the target gene include those against the target gene-specific microRNA. Examples include siRNA and antisense to the target gene.

The formulation form and administration method of the cell growth inhibitor or growth promoter are the same as those for diagnostic agents and therapeutic agents containing nucleic acids such as microRNA and microRNA precursors described later in 10.
10. Diagnostic and therapeutic agents that contain nucleic acids such as microRNA and microRNA precursors Nucleic acids such as microRNA and microRNA precursors control the expression of genes that have target sequences or control the expression of nucleic acids such as microRNAs By controlling, it can be used as a therapeutic agent for diseases caused by abnormal growth of cells such as cancer. Furthermore, siRNA against the target gene of the nucleic acid can be used as a therapeutic agent for diseases caused by abnormal cell proliferation or differentiation by controlling the expression of the gene. Diseases caused by abnormal cell proliferation include cancer, arteriosclerosis, rheumatoid arthritis, prostatic hypertrophy, vascular restenosis after percutaneous transvascular coronary angioplasty, pulmonary fibrosis, glomerulonephritis and autoimmunity Diseases can be raised.

In addition, by quantifying nucleic acids or detecting mutations, it is possible to diagnose diseases caused by abnormal proliferation or differentiation of cells such as cancer.
A diagnostic agent containing a nucleic acid is a reagent necessary for quantifying a nucleic acid or detecting a mutation, such as a buffer, a salt, a reaction enzyme, a labeled protein that binds to the nucleic acid, depending on the target diagnostic method, And a coloring agent for detection.

A therapeutic agent containing a nucleic acid as an active ingredient can be administered alone, but usually mixed with one or more pharmacologically acceptable carriers and well known in the pharmaceutical arts. It is desirable to administer it as a pharmaceutical preparation produced by any method.
It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous is desirable. Can be given intravenously.

Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.

  For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles.

Specific examples of the carrier include lactose and glycerin. Depending on the nature of the nucleic acid or microRNA precursor, as well as the carrier used, a formulation such as an aerosol or dry powder is possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 20 mg / kg per day for an adult.

A therapeutic agent containing a nucleic acid as an active ingredient can also be produced by preparing a vector that expresses a nucleic acid and a base used for the nucleic acid therapeutic agent [Nature Genet., 8 , 42 (1994)].
The base used for the therapeutic agent may be any base as long as it is usually used for injections, such as distilled water, sodium chloride or a salt solution such as a mixture of sodium chloride and an inorganic salt, mannitol, lactose, dextran. And a solution such as glucose, an amino acid solution such as glycine and arginine, an organic acid solution or a mixed solution of a salt solution and a glucose solution, and the like. In addition, according to a conventional method, these bases are mixed with an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and soybean oil, or an auxiliary such as a surfactant such as lecithin or a nonionic surfactant. An injection may be prepared as a suspension or dispersion. These injections can be prepared as preparations for dissolution at the time of use by operations such as pulverization and freeze-drying. The therapeutic agent can be used for treatment as it is in the case of a liquid just before the treatment, and in the case of an individual, it can be dissolved in the above sterilized base if necessary.

Examples of the vector for expressing the nucleic acid include the recombinant virus vector virus vector prepared by the method 5 above, and more specifically, a retrovirus vector, a lentivirus vector, and the like.
For example, a virus vector can be prepared by preparing a complex by combining a nucleic acid with a polylysine-conjugated antibody specific for an adenovirus hexon protein, and binding the resulting complex to an adenovirus vector. The virus vector stably reaches the target cell, is taken up into the cell by endosomes, is degraded in the cell, and the nucleic acid can be efficiently expressed.

In addition, viral vectors based on Sendai virus (-) strand RNA virus have also been developed (WO97 / 16538, WO97 / 16539), and using the Sendai virus, a Sendai virus incorporating a nucleic acid can be produced. Can do.
Nucleic acids can also be transferred by non-viral nucleic acid transfer methods. For example, calcium phosphate coprecipitation method [Virology, 52 , 456-467 (1973); Science, 209 , 1414-1422 (1980)], microinjection method [Proc. Natl. Acad. Sci. USA, 77 , 5399-5403 ( Proc. Natl. Acad. Sci. USA, 77 , 7380-7384 (1980); Cell, 27 , 223-231 (1981); Nature, 294 , 92-94 (1981)], liposome-mediated membrane Fusion-mediated transfer [Proc. Natl. Acad. Sci. USA, 84 , 7413-7417 (1987); Biochemistry, 28 , 9508-9514 (1989); J. Biol. Chem., 264 , 12126-12129 (1989) Hum. Gene Ther., 3 , 267-275 (1992); Science, 249 , 1285-1288 (1990); Circulation, 83 , 2007-2011 (1992)] or direct DNA uptake and receptor-mediated DNA transfer [Science, 247 , 1465-1468 (1990); J. Biol. Chem., 266 , 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87 , 3655-3659 (1991); Biol. Chem., 264,
16985-16987 (1989); BioTechniques, 11 , 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87 , 3410-3414 (1990); Proc. Natl. Acad. Sci. USA, 88 , 4255 Proc. Natl. Acad. Sci. USA, 87 , 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88 , 8850-8854 (1991); Hum. Gene Ther., 3 , 147-154 (1991)].

Liposome-mediated membrane fusion-mediated transfer allows the nucleic acid to be taken up and expressed locally by administering the liposome preparation directly to the target tissue [Hum. Gene Ther., 3 , 399 (1992)]. Direct DNA uptake techniques are preferred for targeting DNA directly to a lesion.
For example, receptor-mediated DNA transfer can be performed by binding DNA (usually in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-DNA conjugate can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the DNA-protein complex occurs. In order to prevent intracellular destruction of DNA, adenovirus can be co-infected to disrupt endosomal function.
11. Method for Measuring Growth of Cancer Cells and Other Cells The method for measuring cell growth is not particularly limited as long as it is a method capable of measuring an index reflecting the number of cells and the cell growth rate. Viable cell count measurement, DNA synthesis rate measurement, total protein mass measurement, and the like can be used. An example of a method for evaluating the number of living cells is a method of measuring the amount of ATP in the cells. It is known that the amount of ATP in cells is proportional to the number of cells in culture (J. Immunol. Meth., 160 , 81-88 (1993)). More specific methods for measuring the amount of ATP in cells include the MTT method and the XTT method (J. Immunol. Meth., 65 , 55-63 (1983)). Another example is a method of measuring ATP by luminescence of a luciferin substrate by an ATP-dependent enzyme luciferase. As a kit for measuring the amount of ATP in cells, for example, CellTite-Glo ^R Luminescent Cell Viability Assay (manufactured by Promega) may be used.
12 Methods for measuring the degree of cell death of cancer cells Methods for measuring the degree of cell death include staining dead cells with dyes such as Propium Iodide, and measuring the activity of enzymes that have leaked outside the cell due to cell death. A method or the like can be used. For the latter, for example, a method for measuring the enzyme activity of adenylate kinase leaked out of the cell can be used. More specifically, ToxiLight Non-Destructive Cytotoxicity BioAssay Kit (manufactured by Lonza) may be used.

The degree of apoptosis can also be measured by limiting to apoptosis (programmed cell death) which is particularly important in relation to cancer. Methods for assessing cell apoptosis include measuring the degree of DNA fragmentation, measuring changes in cell membrane lipids, and caspase 3/7, a protease in the cell induced by apoptosis. A method for measuring the activity can be mentioned. A more specific method for measuring caspase 3/7 activity in cells is a method of measuring luciferin substrate released by caspase 3/7 activity by luciferin luminescence by luciferase enzyme reaction. As a kit for measuring caspase 3/7 activity in cells, for example, Caspase-Glo ^R 3/7 Assay (manufactured by Promega) may be used.

  The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.

Viable cell rate and apoptotic activity in colon cancer cell lines in which microRNAs are forcibly expressed
Pre-miR ^™ miRNA Precursor Molecules (manufactured by Ambion) was introduced into a colon cancer-derived cell line, and the influence of microRNAs produced from the molecule on the viable cell rate and apoptotic activity was examined.
The DLD-1 human colorectal cancer-derived cell line (hereinafter referred to as DLD-1) was obtained from the American Type Culture Collection (hereinafter referred to as ATCC) and contains 10% fetal bovine serum (FBS, manufactured by JRH Biosciences) RPMI1640 The cells were cultured in a medium (manufactured by Invitrogen) in an incubator at 37 ° C. with 5% CO ₂ concentration.

DLD-1 was seeded in a 96-well plate at 2500 per well, and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-7 (SEQ ID NO: 3), 15a, 15b, 16, 18a (SEQ ID NO: 5), 18a * (SEQ ID NO: 7), 28 (SEQ ID NO: 17), 95 (SEQ ID NO: 24), 124a , 132 (SEQ ID NO: 29), 133a (SEQ ID NO: 31), 133b (SEQ ID NO: 32), 147 (SEQ ID NO: 37), 181b, 181c (SEQ ID NO: 39), 181d (SEQ ID NO: 40), 184 (SEQ ID NO: 43) ), 192 (SEQ ID NO: 44), 193a (SEQ ID NO: 46), 193b (SEQ ID NO: 47), 195 (SEQ ID NO: 48), 196a (SEQ ID NO: 51), 196b (SEQ ID NO: 52), 197 (SEQ ID NO: 53) , 202 (SEQ ID NO: 56), 204 (SEQ ID NO: 57), 211 (SEQ ID NO: 58), 212 (SEQ ID NO: 30), 215 (SEQ ID NO: 45), 299-3p (SEQ ID NO: 62), 299-5p (sequence) No. 63), 324-3p (SEQ ID NO: 66), 337 (SEQ ID NO: 70), 342 (SEQ ID NO: 72), 362 (SEQ ID NO: 74), 368 (SEQ ID NO: 75), 376a (SEQ ID NO: 77), 376b ( SEQ ID NO: 78), 380-5p (SEQ ID NO: 81), 422a (SEQ ID NO: 84), 422b (SEQ ID NO: 85), 424 (SEQ ID NO: 49), 432 * (SEQ ID NO: 88), 452 (SEQ ID NO: 92), 452 * (SEQ ID NO: 93), 455 (SEQ ID NO: 94), 483 (SEQ ID NO: 95), 489 (SEQ ID NO: 98), 491 (SEQ ID NO: 99), 493-5p (arrangement) (Column number 100), 494 (sequence number 101), 497 (sequence number 50), 501 (sequence number 102), 504 (sequence number 103), 506 (sequence number 105), 512-3p (sequence number 110), 512 -5p (SEQ ID NO: 111), 517 * (SEQ ID NO: 114), 517b (SEQ ID NO: 117), 517c (SEQ ID NO: 116), 518b (SEQ ID NO: 118), 518c * (SEQ ID NO: 121), 518d (SEQ ID NO: 120) ), 523 (SEQ ID NO: 125), 527 (SEQ ID NO: 129), let-7g, let-7i Pre-miR ^TM miRNA Precursor Molecules, lipofection method, specifically Lipofectamine2000 (Invitrogen) Depending on the method used, DLD-1 was introduced to a final concentration of 5 nM or 25 nM. Pre-miR / miRNA Precursor Molecules-Negative Control # 2 (hereinafter referred to as miR-negacon # 2) (manufactured by Ambion) was also introduced into DLD-1 as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after the introduction of the molecule by the lipofection method, the cell rate was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. The relative viable cell ratio of each control group (Lipofectamine 2000 only) was calculated assuming that the viable cell ratio of DLD-1 was 1.0. As a result, as shown in Table 5, hsa-miR-7, 28, 95, 147, 181d, 192, 193a, 193b, 195, 196a, 197, 211, 215, 299-3p, 299-5p, 337 , 342, 362, 368, 376a, 376b, 380-5p, 422b, 432 *, 452, 483, 489, 491, 493-5p, 494, 501, 506, 517c, 518b, 518d As a result, a decrease in the viable cell rate of 40% or more was observed. By introducing a molecule that produces hsa-miR-15b, 124a, which is known to be associated with cancer, a decrease in the viable cell rate of 40% or more was observed.

またリポフェクション法により該分子（終濃度25ｎM）を導入した2日後に、Caspase-Glo^R3/7 assay（Promega社製）を用いて製品に添付された説明書に記載された方法に従いカスパーゼ3/7活性を測定した。対照区（Lipofectamine2000のみ）のDLD-1のカスパーゼ3/7活性値を1.0として、それぞれの相対カスパーゼ3/7活性値を計算した。その結果、表６に示したように、hsa-miR-18a, 18a*, 132, 133a, 133b, 181c, 181d, 184, 192, 193a, 195, 196a, 196b, 197, 202, 204, 211, 212, 215, 299-3p, 299-5p, 324-3p, 337, 342, 362, 368, 376a, 380-5p, 422a, 422b, 424, 432*, 452*, 455, 483, 491, 493-5p, 497, 501, 504, 512-3p, 512-5p, 517*, 517b, 517c, 518c*, 523, 527を生成する分子導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。癌との関連が知られているhsa-miR-15a, 15b, 16, 181b, let-7g, let-7iを生成する分子を導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。

In addition, two days after the introduction of the molecule (final concentration 25 nM) by the lipofection method, the caspase 3/3 according to the method described in the instructions attached to the product using Caspase-Glo ^R 3/7 assay (manufactured by Promega). 7 activities were measured. The relative caspase 3/7 activity value of each control group (Lipofectamine 2000 only) was calculated by setting the caspase 3/7 activity value of DLD-1 to 1.0. As a result, as shown in Table 6, hsa-miR-18a, 18a *, 132, 133a, 133b, 181c, 181d, 184, 192, 193a, 195, 196a, 196b, 197, 202, 204, 211, 212, 215, 299-3p, 299-5p, 324-3p, 337, 342, 362, 368, 376a, 380-5p, 422a, 422b, 424, 432 *, 452 *, 455, 483, 491, 493- By introducing molecules that generate 5p, 497, 501, 504, 512-3p, 512-5p, 517 *, 517b, 517c, 518c *, 523, 527, caspase 3/7 activity increased by more than 50% Admitted. Introduction of molecules that produce hsa-miR-15a, 15b, 16, 181b, let-7g, let-7i, which are known to be associated with cancer, increased caspase 3/7 activity by more than 50%. Admitted.

別の試験として、DLD-1を96穴プレートに1穴あたり2500個になるように播種し、10％ FBSを含むRPMI培地で一晩培養した。１日後、hsa-miR-7(配列番号3), 24(配列番号11), 28(配列番号17), 34c, 95(配列番号24), 124a, 132(配列番号29), 134(配列番号33), 142-3p(配列番号35), 147(配列番号37), 181a, 182*(配列番号41), 183(配列番号42), 192(配列番号44), 197(配列番号53), 211(配列番号58), 222(配列番号61), 299-3p(配列番号62), 329(配列番号68), 337(配列番号70), 342(配列番号72), 362(配列番号74), 368(配列番号75), 376a(配列番号77), 376b(配列番号78), 377(配列番号79), 380-5p(配列番号81), 422b(配列番号85), 424(配列番号49), 431(配列番号87), 432*(配列番号88), 452(配列番号92), 452*(配列番号93), 455(配列番号94), 483(配列番号95), 485-5p(配列番号96), 489(配列番号98), 491(配列番号99), 493-5p(配列番号100), 494(配列番号101), 505(配列番号104), 506(配列番号105), 509(配列番号107), 510(配列番号108), 517a(配列番号115), 517c(配列番号116)を生成するPre-miR^TMmiRNA Precursor Molecules（Ambion社製）を、リポフェクション法、具体的には、Lipofectamine2000（Invitrogen社製）を用いた方法により、終濃度が50nMとなるようDLD-1に導入した。用いた。また、miR-negacon#2（Ambion社製）もDLD-1に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

As another test, DLD-1 was seeded in a 96-well plate at 2500 cells per well and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-7 (SEQ ID NO: 3), 24 (SEQ ID NO: 11), 28 (SEQ ID NO: 17), 34c, 95 (SEQ ID NO: 24), 124a, 132 (SEQ ID NO: 29), 134 (SEQ ID NO: 33), 142-3p (SEQ ID NO: 35), 147 (SEQ ID NO: 37), 181a, 182 * (SEQ ID NO: 41), 183 (SEQ ID NO: 42), 192 (SEQ ID NO: 44), 197 (SEQ ID NO: 53), 211 (SEQ ID NO: 58), 222 (SEQ ID NO: 61), 299-3p (SEQ ID NO: 62), 329 (SEQ ID NO: 68), 337 (SEQ ID NO: 70), 342 (SEQ ID NO: 72), 362 (SEQ ID NO: 74) , 368 (SEQ ID NO: 75), 376a (SEQ ID NO: 77), 376b (SEQ ID NO: 78), 377 (SEQ ID NO: 79), 380-5p (SEQ ID NO: 81), 422b (SEQ ID NO: 85), 424 (SEQ ID NO: 49) ), 431 (SEQ ID NO: 87), 432 * (SEQ ID NO: 88), 452 (SEQ ID NO: 92), 452 * (SEQ ID NO: 93), 455 (SEQ ID NO: 94), 483 (SEQ ID NO: 95), 485-5p ( SEQ ID NO: 96), 489 (SEQ ID NO: 98), 491 (SEQ ID NO: 99), 493-5p (SEQ ID NO: 100), 494 (SEQ ID NO: 101), 505 (SEQ ID NO: 104), 506 (SEQ ID NO: 105), 509 (SEQ ID NO: 107), 510 (SEQ ID NO: 108), 517a (SEQ ID NO: ^{115), Pre-miR TM miRNA} Precu for generating 517c (SEQ ID NO: 116) rsor Molecules (Ambion) was introduced into DLD-1 to a final concentration of 50 nM by a lipofection method, specifically, a method using Lipofectamine 2000 (Invitrogen). Using. MiR-negacon # 2 (Ambion) was also introduced into DLD-1 as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three or five days after the introduction of the molecule by the lipofection method, the cell rate was measured using the CellTiter-Glo ^™ Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated assuming that the viable cell ratio of DLD-1 in the miR-negacon # 2-introduced section was 1.0. As a result, as shown in Table 7, hsa-miR-7, 24, 28, 95, 132, 134, 142-3p, 147, 182 *, 183, 192, 197, 211, 222, 299-3p, 329, 337, 342, 362, 368, 376a, 376b, 377, 380-5p, 422b, 424, 431, 432 *, 452, 452 *, 455, 483, 485-5p, 489, 491, 493-5p, By introducing molecules that generate 494, 505, 506, 509, 510, 517a, and 517c, a decrease in live cell activity of 50% or more was observed. In addition, the introduction of a molecule that produces hsa-miR-34c, 181a, 124a, which is known to be associated with cancer, showed a decrease in the viable cell rate of 50% or more.

また、マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドをリポフェクション法を用いて、終濃度が50nMとなるようDLD-1に導入した。マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドとしては、hsa-miR-197(配列番号53), 483(配列番号95)のAnti-miR^TM miRNA Inhibitors（Ambion社製）を用いた。Anti-miR・miRNA Inhibitors-Negative Control #1（以下、anti-miR-negacon#1と称す）（Ambion社製）もDLD-1に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

In addition, antisense oligonucleotides complementary to microRNA were introduced into DLD-1 to a final concentration of 50 nM using the lipofection method. Anti-miR ^™ miRNA Inhibitors (manufactured by Ambion) of hsa-miR-197 (SEQ ID NO: 53) and 483 (SEQ ID NO: 95) were used as antisense oligonucleotides that complement microRNA. Anti-miR / miRNA Inhibitors-Negative Control # 1 (hereinafter referred to as anti-miR-negacon # 1) (Ambion) was also introduced into DLD-1 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after introduction of the antisense oligonucleotide by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated by setting the viable cell ratio of DLD-1 in the anti-miR-negacon # 1 introduction group to 1.0. As a result, as shown in Table 8, by introducing an antisense oligonucleotide complementary to hsa-miR-197, 483, a decrease in the viable cell rate of 50% or more was observed.

Viable cell rate and apoptotic activity in ovarian cancer cell lines in which microRNAs are forcibly expressed
Pre-miR ^™ miRNA Precursor Molecules (manufactured by Ambion) was introduced into an ovarian cancer-derived cell line, and the influence of microRNA generated from the molecule on the viable cell rate and apoptotic activity was examined.
A2780 human ovarian carcinoma-derived cell line (Nature, 295, 116-119, ( 1982); Science, 224, 994-996, (1984);.. Semin Oncol, 11, 285-298 (1984); hereinafter, the A2780 Was cultured in RPMI1640 medium (Invitrogen) containing 5% FBS (manufactured by JRH Biosciences) in an incubator at 37 ° C. and 5% CO ₂ concentration.

A2780 was seeded in a 96-well plate at 2500 per well and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-1 (SEQ ID NO: 1), 7 (SEQ ID NO: 3), 15a, 15b, 16, 18a (SEQ ID NO: 5), 18a * (SEQ ID NO: 7), 18b (SEQ ID NO: 6), 28 (SEQ ID NO: 17), 34b, 95 (SEQ ID NO: 24), 98 (SEQ ID NO: 25), 124a, 133a (SEQ ID NO: 31), 133b (SEQ ID NO: 32), 143, 145, 147 (SEQ ID NO: 37), 181c (SEQ ID NO: 39), 192 (SEQ ID NO: 44), 193a (SEQ ID NO: 46), 193b (SEQ ID NO: 47), 195 (SEQ ID NO: 48), 196a (SEQ ID NO: 51), 196b (SEQ ID NO: 52), 197 ( (SEQ ID NO: 53), 199a * (SEQ ID NO: 54), 202 (SEQ ID NO: 56), 204 (SEQ ID NO: 57), 211 (SEQ ID NO: 58), 212 (SEQ ID NO: 30), 215 (SEQ ID NO: 45), 299- 3p (SEQ ID NO: 62), 324-3p (SEQ ID NO: 66), 324-5p (SEQ ID NO: 67), 337 (SEQ ID NO: 70), 342 (SEQ ID NO: 72), 346 (SEQ ID NO: 73), 362 (SEQ ID NO: 74), 368 (SEQ ID NO: 75), 376a (SEQ ID NO: 77), 376b (SEQ ID NO: 78), 378 (SEQ ID NO: 80), 380-5p (SEQ ID NO: 81), 382 (SEQ ID NO: 82), 422a (sequence) No. 84), 422b (SEQ ID NO: 85), 424 (SEQ ID NO: 49), 432 * (SEQ ID NO: 88), 451 (SEQ ID NO: 91), 452 (SEQ ID NO: 92), 452 * ( (Column number 93), 455 (SEQ ID NO: 94), 483 (SEQ ID NO: 95), 489 (SEQ ID NO: 98), 491 (SEQ ID NO: 99), 493-5p (SEQ ID NO: 100), 494 (SEQ ID NO: 101), 497 (SEQ ID NO: 50), 501 (SEQ ID NO: 102), 504 (SEQ ID NO: 103), 506 (SEQ ID NO: 105), 511 (SEQ ID NO: 109), 512-5p (SEQ ID NO: 111), 517a (SEQ ID NO: 115), 517c (SEQ ID NO: 116), 518b (SEQ ID NO: 118), 518c (SEQ ID NO: 119), 518c * (SEQ ID NO: 121), 518d (SEQ ID NO: 120), 527 (SEQ ID NO: 129), let-7a, let-7b , let-7d, let-7d, let-7e, let-7f, let-7g, let-7i, Pre-miR ^TM miRNA Precursor Molecules, Lipofectamine 2000 (manufactured by Invitrogen) Was introduced into A2780 so that the final concentration was 5 nM or 25 nM. MiR-negacon # 2 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after the introduction of the molecule by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. The relative viable cell ratio was calculated assuming that the viable cell ratio of A2780 in the control group (Lipofectamine 2000 only) was 1.0. As a result, as shown in Table 9 (Table 9-1 and Table 9-2), hsa-miR-1, 7, 18a, 18a *, 18b, 28, 95, 98, 133a, 133b, 147, 181c , 192, 193a, 193b, 195, 196a, 196b, 197, 199a *, 202, 204, 211, 212, 215, 299-3p, 324-3p, 324-5p, 342, 346, 362, 368, 376a, 376b, 378, 380-5p, 382, 422a, 422b, 424, 432 *, 451, 452, 452 *, 455, 483, 489, 491, 493-5p, 494, 497, 501, 504, 506, 511, By introducing a molecule that produces 512-5p, 517a, 517c, 518b, 518c, 518c *, 518d, 527, a decrease in live cell activity of 40% or more was observed. In addition, hsa-miR-15a, 15b, 16, 34b, 124a, 143, 145, let-7a, let-7b, let-7d, let-7e, let-7f, let By introducing molecules that produce -7g and let-7i, a decrease in the percentage of viable cells of 40% or more was observed.

またリポフェクション法により該分子（終濃度25ｎM）を導入した2日後に、Caspase-Glo^R3/7 assay（Promega社製）を用いて製品に添付された説明書に記載された方法に従いカスパーゼ3/7活性を測定した。対照区（Lipofectamine2000のみ）のA2780のカスパーゼ3/7活性値を1.0として、それぞれの相対カスパーゼ3/7活性値を計算した。その結果、表１０に示したようにhsa-miR-18a*, 133a, 193a, 193b, 197, 199a*, 212, 324-3p, 337, 432*, 452*, 489, 491, 493-5p, 512-5p, 517a, 517c, 527を生成する分子を導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。また、癌との関連が知られているhsa-miR-124aを生成する分子を導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。

In addition, two days after the introduction of the molecule (final concentration 25 nM) by the lipofection method, the caspase 3/3 according to the method described in the instructions attached to the product using Caspase-Glo ^R 3/7 assay (manufactured by Promega). 7 activities were measured. The relative caspase 3/7 activity value was calculated by setting the caspase 3/7 activity value of A2780 in the control group (Lipofectamine 2000 only) to 1.0. As a result, as shown in Table 10, hsa-miR-18a *, 133a, 193a, 193b, 197, 199a *, 212, 324-3p, 337, 432 *, 452 *, 489, 491, 493-5p, By introducing molecules that generate 512-5p, 517a, 517c, 527, an increase in caspase 3/7 activity of 50% or more was observed. Furthermore, by introducing a molecule that produces hsa-miR-124a, which is known to be associated with cancer, an increase in caspase 3/7 activity of 50% or more was observed.

別の試験として、A2780を96穴プレートに1穴あたり2500個になるように播種し、10％FBSを含むRPMI培地で一晩培養した。１日後、、hsa-miR-7(配列番号3), 15a, 15b, 18a*(配列番号7), 24(配列番号11), 26a(配列番号14), 26b(配列番号15), 34a, 34b, 34c, 124a, 130a(配列番号26), 130b(配列番号27), 134(配列番号33), 147(配列番号37), 192(配列番号44), 193a(配列番号46), 193b(配列番号47), 196a(配列番号51), 196b(配列番号52), 197(配列番号53), 199a*(配列番号54), 204(配列番号57), 206(配列番号2), 211(配列番号58), 212(配列番号30), 215(配列番号45), 301(配列番号28), 346(配列番号73), 368(配列番号75), 371(配列番号76), 376b(配列番号78), 378(配列番号80), 422a(配列番号84), 424(配列番号49), 431(配列番号87), 432*(配列番号88), 433(配列番号89), 449(配列番号90), 452(配列番号92), 483(配列番号95), 485-5p(配列番号96), 488(配列番号97), 489(配列番号98), 491(配列番号99), 493-5p(配列番号100), 497(配列番号50), 504(配列番号103), 505(配列番号104), 506(配列番号105), 507(配列番号106), 510(配列番号108), 511(配列番号109), 512-5p(配列番号111), 513(配列番号112), 517a(配列番号115), 517c(配列番号116), 518b(配列番号118), 518c(配列番号119), 518d(配列番号120), 526a(配列番号128), 527(配列番号129), let-7b, let-7c, let-7iのPre-miR^TMmiRNA Precursor Moleculesを、リポフェクション法、具体的には、Lipofectamine2000（Invitrogen社製）を用いて、終濃度が50nMとなるようA2780に導入した。また、miR-negacon#2（Ambion社製）もA2780に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

As another test, A2780 was seeded in a 96-well plate at 2500 cells per well and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-7 (SEQ ID NO: 3), 15a, 15b, 18a * (SEQ ID NO: 7), 24 (SEQ ID NO: 11), 26a (SEQ ID NO: 14), 26b (SEQ ID NO: 15), 34a, 34b, 34c, 124a, 130a (SEQ ID NO: 26), 130b (SEQ ID NO: 27), 134 (SEQ ID NO: 33), 147 (SEQ ID NO: 37), 192 (SEQ ID NO: 44), 193a (SEQ ID NO: 46), 193b ( (SEQ ID NO: 47), 196a (SEQ ID NO: 51), 196b (SEQ ID NO: 52), 197 (SEQ ID NO: 53), 199a * (SEQ ID NO: 54), 204 (SEQ ID NO: 57), 206 (SEQ ID NO: 2), 211 ( (SEQ ID NO: 58), 212 (SEQ ID NO: 30), 215 (SEQ ID NO: 45), 301 (SEQ ID NO: 28), 346 (SEQ ID NO: 73), 368 (SEQ ID NO: 75), 371 (SEQ ID NO: 76), 376b (sequence) 78), 378 (SEQ ID NO: 80), 422a (SEQ ID NO: 84), 424 (SEQ ID NO: 49), 431 (SEQ ID NO: 87), 432 * (SEQ ID NO: 88), 433 (SEQ ID NO: 89), 449 (sequence) No. 90), 452 (SEQ ID NO: 92), 483 (SEQ ID NO: 95), 485-5p (SEQ ID NO: 96), 488 (SEQ ID NO: 97), 489 (SEQ ID NO: 98), 491 (SEQ ID NO: 99), 493- 5p (SEQ ID NO: 100), 497 (SEQ ID NO: 50), 504 (SEQ ID NO: 103), 505 (SEQ ID NO: 104), 506 (SEQ ID NO: 105), 507 ( (Column number 106), 510 (sequence number 108), 511 (sequence number 109), 512-5p (sequence number 111), 513 (sequence number 112), 517a (sequence number 115), 517c (sequence number 116), 518b (SEQ ID NO: 118), 518c (SEQ ID NO: 119), 518d (SEQ ID NO: 120), 526a (SEQ ID NO: 128), 527 (SEQ ID NO: 129), let-7b, let-7c, let-7i Pre-miR ^TM miRNA Precursor Molecules was introduced into A2780 to a final concentration of 50 nM using a lipofection method, specifically, Lipofectamine 2000 (Invitrogen). MiR-negacon # 2 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three or five days after the introduction of the molecule by the lipofection method, the cell viability was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. The relative viable cell ratio was calculated assuming that the viable cell ratio of A2780 in the miR-negacon # 2-introduced zone was 1.0. As a result, as shown in Table 11 (Table 11-1 and Table 11-2), hsa-miR-7, 18a *, 24, 26a, 26b, 134, 147, 192, 193a, 193b, 196a, 196b , 197, 199a *, 204, 206, 211, 212, 215, 346, 368, 371, 376b, 378, 422a, 424, 431, 432 *, 433, 449, 452, 483, 485-5p, 488, 489 , 491, 493-5p, 497, 504, 505, 506, 507, 510, 511, 512-5p, 513, 517a, 517c, 518b, 518c, 518d, 526a, 527 A decrease in the viable cell rate of 50% or more was observed. In addition, by introducing microRNA precursors of hsa-miR-15a, 15b, 34a, 34b, 34c, 124a, let-7b, let-7c, let-7i, which are known to be associated with cancer, 50 A decrease in the viable cell rate of more than 1% was observed. On the contrary, by introducing a molecule that generates hsa-miR-130a, 130b, 301, an increase in the viable cell rate was observed.

また、マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドを、リポフェクション法を用いて、終濃度が50nMとなるようA2780に導入した。マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドとしては、hsa-miR-10a(配列番号4), 20b(配列番号8), 25(配列番号12), 27b(配列番号16), 29a(配列番号18), 29c(配列番号19), 30b(配列番号20), 30c(配列番号21), 32(配列番号13), 93(配列番号22), 106a(配列番号9), 106b(配列番号10), 137(配列番号34), 146a(配列番号36), 149(配列番号38), 191, 195(配列番号48), 200a(配列番号55), 204(配列番号57), 205(配列番号59), 208(配列番号60), 212(配列番号30), 215(配列番号45), 301(配列番号28), 302a*(配列番号64), 302b*(配列番号65), 302d(配列番号23), 324-3p(配列番号66), 331(配列番号69), 340(配列番号71), 362(配列番号74), 412(配列番号83), 423(配列番号86), 451(配列番号91), 452(配列番号92), 483(配列番号95), 516-5p(配列番号113), 518e(配列番号122), 518f(配列番号123), 520h(配列番号124), 524*(配列番号126), 525(配列番号127)のAnti-miR^TM miRNA Inhibitors（Ambion社製）を用いた。anti-miR-negacon#1（Ambion社製）もA2780に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

Furthermore, antisense oligonucleotides complementary to microRNA were introduced into A2780 using a lipofection method so that the final concentration was 50 nM. Antisense oligonucleotides complementary to microRNA include hsa-miR-10a (SEQ ID NO: 4), 20b (SEQ ID NO: 8), 25 (SEQ ID NO: 12), 27b (SEQ ID NO: 16), 29a (SEQ ID NO: 18) , 29c (SEQ ID NO: 19), 30b (SEQ ID NO: 20), 30c (SEQ ID NO: 21), 32 (SEQ ID NO: 13), 93 (SEQ ID NO: 22), 106a (SEQ ID NO: 9), 106b (SEQ ID NO: 10), 137 (SEQ ID NO: 34), 146a (SEQ ID NO: 36), 149 (SEQ ID NO: 38), 191, 195 (SEQ ID NO: 48), 200a (SEQ ID NO: 55), 204 (SEQ ID NO: 57), 205 (SEQ ID NO: 59) , 208 (SEQ ID NO: 60), 212 (SEQ ID NO: 30), 215 (SEQ ID NO: 45), 301 (SEQ ID NO: 28), 302a * (SEQ ID NO: 64), 302b * (SEQ ID NO: 65), 302d (SEQ ID NO: 23) ), 324-3p (SEQ ID NO: 66), 331 (SEQ ID NO: 69), 340 (SEQ ID NO: 71), 362 (SEQ ID NO: 74), 412 (SEQ ID NO: 83), 423 (SEQ ID NO: 86), 451 (SEQ ID NO: 91), 452 (SEQ ID NO: 92), 483 (SEQ ID NO: 95), 516-5p (SEQ ID NO: 113), 518e (SEQ ID NO: 122), 518f (SEQ ID NO: 123), 520h (SEQ ID NO: 124), 524 * ( SEQ ID NO: 126), 525 (SEQ ID NO: 127) Anti-miR ^{T M} miRNA Inhibitors (Ambion) were used. anti-miR-negacon # 1 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after introduction of the antisense oligonucleotide by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated assuming that the viable cell ratio of A2780 in the anti-miR-negacon # 1 introduction group was 1.0. As a result, as shown in Table 12, hsa-miR-10a, 20b, 25, 27b, 29a, 29c, 30b, 30c, 32, 93, 106a, 106b, 137, 146a, 149, 191, 195, 200a , 204, 205, 208, 212, 215, 301, 302a *, 302b *, 302d, 324-3p, 331, 340, 362, 412, 423, 451, 452, 483, 516-5p, 518e, 518f, 520h , 524 *, 525, the introduction of antisense oligonucleotides was found to reduce the viable cell rate by more than 40%.

Live cell rate and apoptotic activity in prostate cancer-derived cell lines in which microRNAs are forcibly expressed
Pre-miR ^™ miRNA Precursor Molecules (manufactured by Ambion) was introduced into a prostate cancer-derived cell line, and the influence of microRNA generated from the molecule on the viable cell rate and apoptotic activity was examined.

PC-3 human prostate cancer-derived cell line (hereinafter referred to as PC-3) was obtained from ATCC and F-12 Kaighn's medium (Invitrogen) containing 10% fetal bovine serum (FBS) (JRH Biosciences) and cultured in 5% CO ₂ concentration of the incubator in 37 ° C..
PC-3 was seeded in a 96-well plate at 3000 cells per well, and cultured overnight in F-12 Kaighn's medium containing 10% FBS. One day later, hsa-miR-15a, 15b, 16, 18a * (SEQ ID NO: 7), 34b, 124a, 133b (SEQ ID NO: 32), 181b, 192 (SEQ ID NO: 44), 195 (SEQ ID NO: 48), 196b ( SEQ ID NO: 52), 324-3p (SEQ ID NO: 66), 337 (SEQ ID NO: 70), 362 (SEQ ID NO: 74), 368 (SEQ ID NO: 75), 376b (SEQ ID NO: 78), 380-5p (SEQ ID NO: 81) , 432 * (SEQ ID NO: 88), 452 (SEQ ID NO: 92), 452 * (SEQ ID NO: 93), 489 (SEQ ID NO: 98), 491 (SEQ ID NO: 99), 493-5p (SEQ ID NO: 100), 494 (sequence) Pre-miR ^™ miRNA Precursor Molecules producing No. 101) was introduced into PC-3 using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration of 25 nM. MiR-negacon # 2 (Ambion) was also introduced into PC-3 as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after the introduction of the molecule by the lipofection method, the cell rate was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. The relative viable cell ratio of each control group (Lipofectamine 2000 only) was calculated assuming that the viable cell ratio of PC-3 was 1.0. As a result, as shown in Table 13, hsa-miR-18a *, 133b, 192, 195, 196b, 324-3p, 337, 362, 368, 376b, 380-5p, 432 *, 452, 452 *, By introducing molecules that generate 489, 491, 493-5p, 494, a decrease in the viable cell rate of 40% or more was observed. In addition, the introduction of a molecule that produces hsa-miR-15a, 15b, 16, 34b, 124a, 181b, which is known to be associated with cancer, showed a decrease in the viable cell rate of 40% or more.

またリポフェクション法により該分子を導入した2日後に、Caspase-Glo^R 3/7 assay（Promega社製）を用いて製品に添付された説明書に記載された方法に従いカスパーゼ3/7活性を測定した。対照区（Lipofectamine2000のみ）のPC-3のカスパーゼ3/7活性値を1.0として、それぞれの相対カスパーゼ3/7活性値を計算した。その結果、表１４に示したように、hsa-miR-133b, 337, 362, 432*, 452*, 491, 493-5pを生成する分子を導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。また、癌との関連が知られているhsa-miR-181bを生成する分子を導入することにより、50％以上のカスパーゼ3/7活性の増加が認められた。

In addition, 2 days after the introduction of the molecule by lipofection, caspase 3/7 activity was measured using the Caspase-Glo ^R 3/7 assay (Promega) according to the method described in the instructions attached to the product. . The relative caspase 3/7 activity value was calculated by setting the caspase 3/7 activity value of PC-3 in the control group (Lipofectamine 2000 only) to 1.0. As a result, as shown in Table 14, by introducing molecules that generate hsa-miR-133b, 337, 362, 432 *, 452 *, 491, 493-5p, 50% or more of caspase 3/7 Increased activity was observed. In addition, by introducing a molecule that produces hsa-miR-181b, which is known to be associated with cancer, an increase in caspase 3/7 activity of 50% or more was observed.

BrdU uptake activity in colon cancer-derived cell line (DLD-1) forcibly expressing microRNA
Pre-miR ^™ miRNA Precursor Molecules (Ambion) was introduced into DLD-1, and the influence of microRNA generated from the molecule on BrdU incorporation activity was examined.
DLD-1 was seeded in a 96-well plate at 2500 per well, and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-147 (SEQ ID NO: 37), 342 (SEQ ID NO: 72), 362 (SEQ ID NO: 74), 376b (SEQ ID NO: 78), 483 (SEQ ID NO: 95), 494 (SEQ ID NO: 101) are generated. Pre-miR ^™ miRNA Precursor Molecules were introduced into DLD-1 using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration of 25 nM. MiR-negacon # 2 (Ambion) was also introduced into DLD-1 as a negative control. Lipofection followed the method described in the instructions attached to the product.

  One day after the introduction of the molecule by the lipofection method, BrdU uptake activity was measured using BrdU labeling & detection kit III (Roche) according to the method described in the instructions attached to the product. The relative BrdU incorporation activity of DLD-1 in the miR-negacon # 2-introduced section was calculated as 1.0, and the relative BrdU incorporation activity was calculated. As a result, as shown in Table 15, by introducing a molecule that generates hsa-miR-147, 342, 362, 376b, 483, 494, a decrease in BrdU incorporation activity of 40% or more was observed.

マイクロＲＮＡを強制発現させた卵巣癌由来細胞株（A2780）におけるBrdU取り込み活性
Pre-miR^TMmiRNA Precursor Molecules（Ambion社製）をA2780に導入し、該分子から生成されるマイクロＲＮＡがBrdU取り込み活性に及ぼす影響を調べた。
A2780を96穴プレートに1穴あたり4000個になるように播種し、10％ FBSを含むRPMI培地で一晩培養した。１日後、hsa-miR-15a, 16, 124a, 147(配列番号37), 192(配列番号44), 193a(配列番号46), 193b(配列番号47), 215(配列番号45), 378(配列番号80), 432*(配列番号88), 506(配列番号105), 518c(配列番号119)のPre-miR^TM miRNA Precursor Moleculesを、リポフェクション法、具体的には、Lipofectamine2000（Invitrogen社製）を用いて、終濃度が25nMとなるようA2780に導入した。また、miR-negacon#2（Ambion社製）もA2780に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

BrdU uptake activity in ovarian cancer-derived cell line (A2780) with forced expression of microRNA
Pre-miR ^™ miRNA Precursor Molecules (Ambion) was introduced into A2780, and the effect of microRNA generated from the molecule on BrdU uptake activity was examined.
A2780 was seeded in a 96-well plate at 4000 cells per well, and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-15a, 16, 124a, 147 (SEQ ID NO: 37), 192 (SEQ ID NO: 44), 193a (SEQ ID NO: 46), 193b (SEQ ID NO: 47), 215 (SEQ ID NO: 45), 378 ( Pre-miR ^™ miRNA Precursor Molecules of SEQ ID NO: 80), 432 * (SEQ ID NO: 88), 506 (SEQ ID NO: 105), and 518c (SEQ ID NO: 119) are lipofection methods, specifically, Lipofectamine 2000 (manufactured by Invitrogen) Was introduced into A2780 so that the final concentration was 25 nM. MiR-negacon # 2 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

  One day after the introduction of the molecule by the lipofection method, BrdU uptake activity was measured using BrdU labeling & detection kit III (Roche) according to the method described in the instructions attached to the product. The relative BrdU incorporation activity of A2780 in the miR-negacon # 2-introduced section was calculated as 1.0, and the relative BrdU incorporation activity was calculated. As a result, as shown in Table 16, by introducing molecules that generate hsa-miR-hsa-miR-147, 192, 193a, 193b, 215, 378, 432 *, 506, 518c, more than 40% Decreased BrdU uptake activity. In addition, by introducing a molecule that produces hsa-miR-15a, 16, 124a, which is known to be associated with cancer, a decrease in BrdU incorporation activity of 40% or more was observed.

Analysis of the expression level of the protein encoded by the target genes of hsa-miR-337, 342, 432 *, 491, 518b hsa-miR-337, 342, 432 which showed DLD-1 growth inhibition and caspase activity in Example 1 Molecules producing *, 491, 518b were introduced into DLD-1, and changes in the amount of protein encoded by the target gene were examined.
Pre-miR ^TM miRNA Precursor Molecules (manufactured by Ambion) that produces hsa-miR-16, 147, 337, 342, 432 *, 491, 518b has been pre-seeded using Lipofectamine 2000 to a final concentration of 25 nM. 1x10 ⁵ DLD-1 were introduced. miR-negacon # 2 was also introduced as a negative control. Moreover, si-Bcl2l1, si-Birc5, si-Bcl9l (manufactured by QIAGEN) were introduced at a final concentration of 25 nM, and used as a positive control. Two days after transfection, the cells were treated with RIPA buffer [50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 1% Nonident P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Protease Inhibitor Cocktail Set III (Calbiochem Product)]. After separation by SDS-PAGE by a conventional method, the amounts of Bcl2l1, Birc5 and Bcl9l proteins were detected by Western blotting. Anti-Bcl2l antibody (manufactured by BD Biosciences) was used for detection of Bcl2l1, anti-Birc5 antibody (manufactured by R & D Systems) was used for detection of Birc5, and anti-Bcl9l antibody (manufactured by Abnova) was used for detection of Bcl9l. . In addition, the amount of alpha-tubulin protein was detected and used as a control for the sample amount. For detection of alpha-tubulin, an anti-alpha-tubulin antibody (manufactured by Sigma) was used.

  As a result, as shown in FIG. 1, DLD-1 forcibly expressing hsa-miR-337, 342, 491 had a decreased amount of Bcl2l1 protein compared to negative control DLD-1, so the Bcl2l1 gene was It was a target gene of hsa-miR-337, 342, 491 and its expression was shown to be regulated. In addition, as shown in FIG. 2, DLD-1 forcibly expressing hsa-miR-337 had a reduced amount of Birc5 protein compared to negative control DLD-1, so that the Birc5 gene was hsa-miR-337. It was shown that its expression is regulated. Further, as shown in FIG. 3, Dcl-1 that forcibly expressed hsa-miR-432 *, 518b had a decreased amount of Bcl9l protein compared to negative control DLD-1, so the Bcl9l gene was -432 *, a target gene of 518b, and its expression was shown to be regulated.

Viable cell rate and apoptotic activity in colon cancer cell lines in which microRNAs are forcibly expressed
DLD-1 was seeded in a 96-well plate at 2500 per well, and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-493-3p (SEQ ID NO: 2061), 542-3p (SEQ ID NO: 2063), 571 (SEQ ID NO: 2076), 579 (SEQ ID NO: 2077), 625 (SEQ ID NO: 2099), 630 (SEQ ID NO: 2101), 634 (SEQ ID NO: 2103), 650 (SEQ ID NO: 2108), 653 the Pre-miR ^TM miRNA Precursor Molecules of generating (SEQ ID NO: 2110), lipofection method, specifically, Lipofectamine2000 the (Invitrogen Corp.) And introduced into DLD-1 to a final concentration of 50 nM. MiR-negacon # 2 (Ambion) was also introduced into DLD-1 as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after the introduction of the microRNA precursor by the lipofection method, the cell rate was measured using the CellTiter-Glo ^™ Luminescent Cell Viability Assay (Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated assuming that the viable cell ratio of DLD-1 in the miR-negacon # 2-introduced section was 1.0. As a result, as shown in Table 17, by introducing molecules that generate hsa-miR-493-3p, 542-3p, 571, 579, 625, 630, 634, 650, 653, more than 50% A decrease in the viable cell rate was observed.

As another test, two days after the introduction of Pre-miR ^™ miRNA Precursor Molecules that produces hsa-miR-634 by the lipofection method, it was attached to the product using the Caspase-Glo ^R 3/7 assay (manufactured by Promega). Caspase 3/7 activity was measured according to the method described in the instructions. Relative caspase activity was calculated with the caspase activity of DLD-1 in the miR-negacon # 2-introduced section as 1.0. As a result, an increase in caspase activity of 50% or more was observed by introducing a molecule that generates hsa-miR-634.

また、マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドを、リポフェクション法を用いて、終濃度が50nMとなるようDLD-1に導入した。マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドとしては、hsa-miR-411(配列番号2057), 425-5p(配列番号2059), 449b(配列番号917), 532(配列番号2062), 548b(配列番号2066), 558(配列番号2072), 589(配列番号2079), 593(配列番号2081), 599(配列番号2083), 615(配列番号2093), 637(配列番号2104), 657(配列番号2112), 769-5p(配列番号2114), 92b(配列番号819)のAnti-miR^TM miRNA Inhibitors（Ambion社製）を用いた。anti-miR-negacon#1（Ambion社製）もDLD-1に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

In addition, antisense oligonucleotide complementary to microRNA was introduced into DLD-1 using lipofection method so that the final concentration was 50 nM. Antisense oligonucleotides complementary to microRNA include hsa-miR-411 (SEQ ID NO: 2057), 425-5p (SEQ ID NO: 2059), 449b (SEQ ID NO: 917), 532 (SEQ ID NO: 2062), 548b (SEQ ID NO: 2066), 558 (SEQ ID NO: 2072), 589 (SEQ ID NO: 2079), 593 (SEQ ID NO: 2081), 599 (SEQ ID NO: 2083), 615 (SEQ ID NO: 2093), 637 (SEQ ID NO: 2104), 657 (SEQ ID NO: 2112) ), 769-5p (SEQ ID NO: 2114), 92b (SEQ ID NO: 819) Anti-miR ^™ miRNA Inhibitors (Ambion) were used. anti-miR-negacon # 1 (Ambion) was also introduced into DLD-1 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after introduction of the antisense oligonucleotide by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated by setting the viable cell ratio of DLD-1 in the anti-miR-negacon # 1 introduction group to 1.0. As a result, as shown in Table 18, complementary to hsa-miR-411, 425-5p, 449b, 532, 548b, 558, 589, 593, 599, 615, 637, 657, 769-5p, 92b By introducing the sense oligonucleotide, a decrease in the viable cell rate of 40% or more was observed.

Viable cell rate in ovarian cancer-derived cell lines in which microRNAs are forced
A2780 was seeded in a 96-well plate at 2500 per well and cultured overnight in RPMI medium containing 10% FBS. One day later, hsa-miR-421 (SEQ ID NO: 2058), 454-5p (SEQ ID NO: 2060), 542-3p (SEQ ID NO: 2063), 545 (SEQ ID NO: 2064), 548a (SEQ ID NO: 2065), 548d (SEQ ID NO: 2067), 550 (SEQ ID NO: 2068), 551b (SEQ ID NO: 2069), 552 (SEQ ID NO: 2070), 553 (SEQ ID NO: 2071), 557 (SEQ ID NO: 925), 561 (SEQ ID NO: 2073), 563 (SEQ ID NO: 916) ), 565 (SEQ ID NO: 2074), 570 (SEQ ID NO: 2075), 571 (SEQ ID NO: 2076), 584 (SEQ ID NO: 2078), 591 (SEQ ID NO: 2080), 595 (SEQ ID NO: 2082), 600 (SEQ ID NO: 2084) , 601 (SEQ ID NO: 2085), 604 (SEQ ID NO: 2086), 605 (SEQ ID NO: 2087), 606 (SEQ ID NO: 2088), 609 (SEQ ID NO: 2089), 610 (SEQ ID NO: 2090), 611 (SEQ ID NO: 2091), 613 (SEQ ID NO: 803), 614 (SEQ ID NO: 2092), 618 (SEQ ID NO: 2096), 621 (SEQ ID NO: 2097), 624 (SEQ ID NO: 2098), 625 (SEQ ID NO: 2099), 627 (SEQ ID NO: 2100), 630 (SEQ ID NO: 2101), 631 (SEQ ID NO: 2102), 634 (SEQ ID NO: 2103), 637 (SEQ ID NO: 2104), 646 (SEQ ID NO: 2105), 647 (SEQ ID NO: 2106), 648 (SEQ ID NO: 2107), 653 ( (SEQ ID NO: 2110), 654 (arrangement No. 2111), 661 and Pre-miR ^TM miRNA Precursor Molecules of generating (SEQ ID NO: 2113), lipofection method, specifically, using Lipofectamine2000 (Invitrogen Corporation), in the A2780 to final concentration of 50nM Introduced. MiR-negacon # 2 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after the introduction of the molecule by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. The relative viable cell ratio was calculated assuming that the viable cell ratio of A2780 in the miR-negacon # 2-introduced zone was 1.0. As a result, as shown in Table 19, hsa-miR-421, 454-5p, 542-3p, 545, 548a, 548d, 550, 551b, 552, 553, 557, 561, 563, 565, 570, 571 , 584, 591, 595, 600, 601, 604, 605, 606, 609, 610, 611, 613, 614, 618, 621, 624, 625, 627, 630, 631, 634, 637, 646, 647, 648 , 653, 654, and 661, the viable cell rate was reduced by 50% or more.

また、マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドをリポフェクション法を用いて、終濃度が50nMとなるようA2780に導入した。マイクロＲＮＡを相補するアンチセンスオリゴヌクレオチドとしては、hsa-miR-611(配列番号2091), 616(配列番号2094), 617(配列番号2095), 651(配列番号2109), 657(配列番号2112) のAnti-miR^TMmiRNA Inhibitors（Ambion社製）を用いた。anti-miR-negacon#1（Ambion社製）もA2780に導入し、陰性コントロールとした。リポフェクションは、製品に添付された説明書に記載された方法に従った。

In addition, antisense oligonucleotides complementary to microRNA were introduced into A2780 using a lipofection method to a final concentration of 50 nM. Antisense oligonucleotides complementary to microRNA include hsa-miR-611 (SEQ ID NO: 2091), 616 (SEQ ID NO: 2094), 617 (SEQ ID NO: 2095), 651 (SEQ ID NO: 2109), 657 (SEQ ID NO: 2112) Anti-miR ^™ miRNA Inhibitors (Ambion) were used. anti-miR-negacon # 1 (Ambion) was also introduced into A2780 and used as a negative control. Lipofection followed the method described in the instructions attached to the product.

Three days after introduction of the antisense oligonucleotide by the lipofection method, the viable cell ratio was measured using CellTiter-Glo ^™ Luminescent Cell Viability Assay (manufactured by Promega) according to the method described in the instructions attached to the product. . The relative viable cell ratio was calculated assuming that the viable cell ratio of A2780 in the anti-miR-negacon # 1 introduction group was 1.0. As a result, as shown in Table 20, by introducing an antisense oligonucleotide complementary to hsa-miR-611, 616, 617, 651, 657, a decrease in the viable cell rate of 40% or more was observed. .

Tumor formation by transplanting nude mice of colon cancer cell lines that forcedly expressed microRNAs Introduced molecules producing hsa-miR-147, 342, 491 that showed DLD-1 growth inhibition and caspase activity in Example 1 DLD-1 was transplanted into nude mice, and the influence of microRNA on tumor formation was examined.
The hsa-miR-147, 342, 491 generates a ^{Pre-miR TM miRNA Precursor Molecules (} Ambion , Inc.), were seeded in advance using the Lipofectoamine2000 to a final concentration of 50 nM 1 × 10 ⁶ pieces of DLD- Introduced in 1. miR-negacon # 2 was also introduced as a negative control. One day after the introduction, the cells were detached with a 0.05% trypsin solution (Gibco). The exfoliated cells and Matrigel (BD Bioscience) were mixed, and 2.5 × 10 ⁵ cells were transplanted subcutaneously to the side of 6-week-old nude mice. The major axis and minor axis of the tumor formed 7, 10, 13, 16 days after transplantation were measured. Tumor size was calculated by major axis x (minor axis) 2 x 0.5. As shown in FIG. 4, the transplantation of DLD-1 forcibly expressing hsa-miR-147, 342 was shown to suppress tumor formation compared to the negative control.

Moreover, as shown in FIG. 5, it was shown that the transplantation of DLD-1 in which hsa-miR-491 was forcibly expressed inhibited tumor formation compared to the negative control.

Cell growth inhibitors or growth promoters, diagnostic or therapeutic agents for diseases caused by cell growth abnormalities, apoptosis inducers, expression inhibitors or expression promoters for target genes of nucleic acids such as microRNA, cell growth inhibition A method or a growth promoting method can be provided.

Sequence Listing Free Text SEQ ID NO: 1399-n is a, c, g or u

Claims (32)

Cell growth inhibitor or growth promoter containing any of the following nucleic acids (a) to (h) as an active ingredient:
(A) Nucleic acid consisting of a base sequence represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315-2321 (b) SEQ ID NOs: 1-939, 2057-2132, 2212-2261 and 2315 A nucleic acid consisting of a base sequence represented by any one of ˜2321, and a nucleic acid of 17 to 28 bases (c) represented by any one of SEQ ID NOs: 1-939, 2057-2132, 2212-2261, and 2315-2321 (D) a nucleic acid comprising a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 939, 2057 to 2132, 2212 to 2261, and 2315 to 2321 Nucleic acids that hybridize with stringent conditions under complementary conditions (e) SEQ ID NOs: 1-939, 2057- 132, 2212 to 2261 and 2315 to 2321. Nucleic acids comprising the second to eighth base sequences of the base sequence represented by any of (2) SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 Nucleic acid consisting of a base sequence represented by any of the above (g) a base having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 Nucleic acid consisting of sequence (h) Nucleic acid that hybridizes under stringent conditions with a complementary strand of nucleic acid consisting of the base sequence represented by any of SEQ ID NOs: 940 to 2056, 2133 to 2211, 2262 to 2314, and 2322 to 2328 The cell growth inhibitor or growth promoter according to claim 1, wherein the nucleic acid is microRNA or a microRNA precursor. A cell growth inhibitor or growth promoter containing, as an active ingredient, a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid according to claim 1. A cell growth inhibitor or growth promoter comprising the vector expressing the nucleic acid according to claim 1 or 3 as an active ingredient. A cell growth inhibitor or growth promoter comprising as an active ingredient a substance that suppresses expression of a gene having the target base sequence of the nucleic acid according to claim 1. A cell growth inhibitor or growth promoter comprising as an active ingredient a substance that promotes the expression of a gene having the target base sequence of the nucleic acid according to claim 1. The cell growth promoter or growth inhibitor according to claim 5 or 6, wherein the substance that suppresses or promotes expression is a nucleic acid. The cell growth promoter or growth inhibitor according to claim 7, wherein the nucleic acid is siRNA. A cell growth inhibitor or growth promoter containing the vector expressing the nucleic acid according to claim 7 as an active ingredient. The therapeutic agent of the disease resulting from the abnormal proliferation of a cell which contains the proliferation inhibitor or proliferation promoter of the cell of any one of Claims 1-9 as an active ingredient. The therapeutic agent of the disease resulting from the abnormal proliferation of a cell which contains the nucleic acid of any one of Claim 1,3 and 7 as an active ingredient. A diagnostic agent for a disease caused by abnormal cell proliferation, comprising as an active ingredient a reagent for detecting the expression level of the nucleic acid according to claim 1, mutation of the nucleic acid, and mutation of a genome encoding the nucleic acid. The therapeutic or diagnostic agent according to any one of claims 10 to 12, wherein the disease caused by abnormal cell proliferation is cancer. A method for treating a disease caused by abnormal cell proliferation, comprising administering an effective amount of the nucleic acid according to any one of claims 1, 3, and 7. A method for diagnosing a disease caused by abnormal cell proliferation, comprising detecting the expression level of the nucleic acid according to claim 1, a mutation of the nucleic acid, and a mutation of a genome encoding the nucleic acid. The treatment or diagnosis method according to claim 14 or 15, wherein the disease caused by abnormal cell proliferation is cancer. Use of the nucleic acid according to any one of claims 1, 3, and 7 for the manufacture of a therapeutic agent for a disease caused by abnormal cell proliferation. The use according to claim 17, wherein the disease caused by abnormal cell proliferation is cancer. An expression inhibitor for a target gene of the nucleic acid, comprising the nucleic acid according to claim 1 as an active ingredient. The expression promoter for the target gene of the nucleic acid according to claim 1, comprising the nucleic acid according to claim 3 as an active ingredient. The expression inhibitor or expression promoter for the target gene of the nucleic acid according to claim 1, comprising the vector according to claim 4 as an active ingredient. A method for promoting cell growth or inhibiting cell growth, comprising using the nucleic acid according to claim 1 or 3. A method for promoting cell growth or inhibiting cell growth, comprising using the vector according to claim 4. A method for promoting cell growth or inhibiting cell growth, comprising using a substance that suppresses expression of a target gene of the nucleic acid according to claim 1. A method for promoting cell growth or inhibiting cell growth, comprising using a substance that promotes expression of a target gene of the nucleic acid according to claim 1. The method for promoting cell proliferation or inhibiting cell proliferation according to claim 24 or 25, wherein the substance that suppresses or promotes expression is a nucleic acid. The method for promoting cell proliferation or inhibiting cell proliferation according to claim 26, wherein the nucleic acid is siRNA. A method for promoting cell growth or inhibiting cell growth using the vector expressing the nucleic acid according to claim 26. A method for suppressing the expression of a target gene of the nucleic acid, wherein the nucleic acid according to claim 1 is used. The method for promoting expression of a target gene of a nucleic acid according to claim 1, wherein the nucleic acid according to claim 3 is used. The method according to claim 1, wherein the vector according to claim 4 is used. A method for screening a cell growth promoter or growth inhibitor, wherein the expression or function of the nucleic acid according to claim 1 is promoted or suppressed.

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